Patent Application
20060269998
The present invention concerns bacteria, particularly probiotic and lactic acid bacteria, and methods and constructs for the control of cell signaling therein.
In the small intestine, lactobacilli represent a major and important component of the commensal microflora. Various Lactobacillus species have been used as probiotic cultures selected for use in foods or dietary adjuncts based on specific benefits exacted from that organism and bioprocessing characteristics desired by the manufacturer. Not all probiotic cultures exhibit the same characteristics, making selection of these strains and clarification of their benefits paramount. Lactic acid bacteria, and lactobacilli specifically, are a common genus included in food products for a range of prospective health-promoting factors.
Lactic acid bacteria (LAB) are naturally found in a variety of environmental niches where they exist as members of complex microbial communities. Survival in each niche depends on the ability of the organism to sense and respond to varying conditions such as temperature, pH, nutrient availability, and cell population density. One major niche that LAB frequently occupy is that of the mammalian gastrointestinal tract (GIT). Multiple signal transduction pathways likely control the expression of adhesion factors, stress response genes, and other genetic determinants that promote survival and competition of LAB within the various compartments of the GIT. Some intestinal lactobacilli, along with Gram-positive and Gram-negative pathogenic bacteria, have the ability to associate with the intestinal epithelium and mucosal layers therein. This association is thought to be important for the realization of certain probiotic properties including competitive exclusion, immunomodulation, and the delivery of biotherapeutics, among others. Although the molecular mechanisms involved with this association are not understood, it is clear that the process is complex, involving host-specific, bacterial-specific, and environmental factors.
Some intestinal lactobacilli, along with Gram-positive and Gram-negative pathogenic bacteria, have the ability to associate with the intestinal epithelium and mucosal layers therein. The ability to colonize these surfaces may afford probiotics a distinct advantage over other indigenous and pathogenic microflora. Of particular interest is the mechanism through which these bacteria survive and potentially colonize the kinetic environment of the intestinal tract. Certain environmental factors have been studied that influence adhesion including pH, growth phase, and the presence of other microorganisms.
Lactobacilli must maintain a balance between meeting their own growth requirements and surviving the hostile conditions, including gastric-acid shock, presented by the host defenses and competing microflora (Tannock (2005) Appl. Environ. Microbiol. 71:8419-8425). However, little is known about how these stressors influence the ability of lactobacilli to associate with the varied substrates in the intestinal environment.
Methods and compositions are provided which improve the adhesion of a bacteria to target substrates and/or improve the stress tolerance of a bacteria. Methods comprise exposing bacteria to adhesion adaptive conditions and thereby increasing the adhesion and/or stress tolerance of the bacteria. Further provided are bacteria having a modulated level of autoinducer-2 (AI-2). Compositions include recombinant bacteria expressing nucleic acid molecules involved in the pathway of autoinducer-2 production. Further provided are autoinducer-2-related fusion proteins, antigenic peptides, antibodies, and vectors. Methods of screening compounds or environmental conditions which will stimulate the production of autoinducer-2 and/or produce an adhesion adaptive response are further provided, as are various methods of use for the bacteria having increased adhesion and/or stress tolerance.
The present invention is explained in further detail in the drawings herein and the specification set forth below.
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
As used herein, “a,” “an” and “the” can be plural or singular as used throughout the specification and claims. For example “a” cell can mean a single cell or a multiplicity of cells.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
I. Overview
Methods and compositions of the invention are provided which improve the adhesion of a bacteria to target substrates and/or improve stress tolerance of a bacteria. Increased adhesion of a commensal organism, such as a probiotic, to cells of the gastrointestinal tract or the urogenital tract find use in reducing the risk of infection of the gut, urogential tract, and wound sites. Similarly, improving the stress tolerance of a probiotic organism, such that the survival rate of the probiotic is increased in the harsh environment of the intestinal tract, will further aid in reducing the risk of infection of the gut, urogential tract, and wound sites.
In one embodiment, the present invention has identified “adhesion adaptive conditions.” As used herein “adhesion adaptive conditions” comprise any physical, chemical, biological, or similar condition that improves the adhesion of bacteria to a substrate. The present invention has demonstrated that preconditioning bacterial cells to these adhesion adaptive conditions results in an increase in adhesion and/or an increase in stress tolerance. As used herein, the improved adhesion properties of the bacteria is referred to as the “adhesion adaptive response” or “AAR.” Polypeptides and polynucleotides whose levels and/or activity are modulated under adhesion adaptive conditions are referenced to herein as “adhesion adaptive response-related” or “AAR-related” polypeptides or polynucleotides. Various AAR-related polynucleotide and polypeptides are disclosed herein. See Tables 8 and 9 below. Additional AAR-related polynucleotides, and their encoded polypeptides, are set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 33, 34, 35, 36, 37, and 38.
Methods and compositions are further provided which modulate the autoinducer-2 pathway of a bacteria, and thereby modulate the adhesion and/or the stress tolerance of the bacteria. More specifically, polynucleotides and polypeptides of the autoinducer-2 (AI-2) production pathway in bacteria are provided. Bacterial cells frequently communicate via quorum sensing mechanisms, which involve the density-dependant recognition of an autoinducer followed by changes in gene expression. One important quorum sensing system that can be used to communicate among and between species is based on a furanosyl borate diester called autoinducer-2 (AI-2), produced in four enzymatic steps from methionine. AI-2 regulates the expression of various phenotypes including virulence factors, DNA processing, cell morphology, motility, biofilm formation, toxin production, light production, and cell division in numerous species (Xavier et al. (2003) Curr. Opin. Microbiol. 6:191-197). Accordingly, methods and compositions are provided which allow for the modulation of the level and/or activity of the polypeptides in the AI-2 pathway and thereby the ability to modulate various bacterial phenotypes is provided. The polynucleotide and polypeptide sequences of the AI-2 pathway are set for in SEQ ID NO:1, 2, 3, 4, 13, 14, 15, 16, 21 or 22. Such sequences are referred to herein as “AI-2-related sequences”.
II. AI-2- Related and AAR-Related Polypeptides and Polynucleotides
Compositions are provided which employ the various AAR-related and AI-2 related polynucleotides and polypeptides of the invention. The invention further provides fragments and variants of these AI-2-related or AAR-related sequences, which can also be used to practice the methods of the present invention. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame, particularly those encoding proteins involved in the production of AI-2 or in the AAR. Isolated nucleic acid molecules of the present invention comprise nucleic acid sequences encoding AI-2-related or AAR-related proteins set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 34, 36, or 38 the nucleic acid sequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 33, 35, or 37 and variants and fragments thereof. The present invention also encompasses antisense nucleic acid molecules, as described below.
Isolated polypeptides and proteins involved in the production of AI-2 or in the AAR, and variants and fragments thereof, are encompassed, as well as methods for producing those polypeptides. For purposes of the present invention, the terms “protein” and “polypeptide” are used interchangeably. Exemplary AI-2-related polypeptides include SEQ ID NOS:2, 4, 14, 16, 22. Exemplary AAR-related polypeptides include SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 34, 36, and 38.
The “production of AI-2” or the “adhesion adapted response (AAR)” refers to a biological or functional activity as determined in vivo or in vitro according to standard assay techniques. For example, the production of AI-2 can be measured using a reporter strain Vibrio harveyi and an autoinducer bioassay (DeKeersmaecker et al. (2003) Microbiology 149:1953-6). Additional assays involve, for example, measuring bacterial survival or growth under the adhesion adaptive conditions described elsewhere herein.
Bacteria and methods of the invention are useful for bacterial fermentations, for example where it is desired to stimulate the production of AI-2, or to promote adhesion or biofilm formation of the bacteria on a substrate for carrying out the fermentative process.
Bacteria of the invention are useful as probiotic bacteria, for example where it is desired to stimulate the production of AI-2, or to promote adhesion or biofilm formation of the bacteria on the gut or intestinal wall to competitively exclude other less desired bacteria therefrom.
Methods of inducing cell signaling or an adhesion adaptive response in bacteria are useful in stimulating the production of AI-2, or in promoting adhesion or biofilm formation of bacteria, including both recombinant and non-recombinant wild type bacteria, to stimulate the production of AI-2, or to promote adhesion or biofilm formation by probiotic bacteria.
The nucleic acid and polypeptide compositions encompassed by the present invention are isolated or substantially purified. By “isolated” or “substantially purified” is intended that the nucleic acid or polypeptide molecules, or biologically active fragments or variants, are substantially or essentially free from components normally found in association with the nucleic acid molecule or protein in its natural state. Such components include other cellular material, culture media from recombinant production, and various chemicals used in chemically synthesizing the proteins or nucleic acids. Preferably, an “isolated” nucleic acid of the present invention is free of nucleic acid sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5′ or 3′ ends). However, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition. For example, in various embodiments, the isolated nucleic acid molecule contains less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequence normally associated with the genomic DNA in the cells from which it was derived. Similarly, a substantially purified protein has less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein, or non-AI-2-related or non-AAR-related protein. When the protein is recombinantly produced, preferably culture medium represents less than 30%, 20%, 10%, or 5% of the volume of the protein preparation, and when the protein is produced chemically, preferably the preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors, or chemicals not related to the production of AI-2 or to the adhesion adaptive conditions.
i. Fragments and Variants
The invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding AI-2-related or AAR-related proteins, as well as the AI-2-related or AAR-related proteins encoded thereby. Fragments and variants of these nucleotide sequences and encoded proteins are also provided. By “fragment” of a nucleotide sequence or protein is intended a portion of the nucleotide or amino acid sequence.
Fragments of the nucleic acid molecules disclosed herein can be used as hybridization probes to identify AI-2-related or AAR-related protein-encoding nucleic acids, or can be used as primers in PCR amplification or mutation of AI-2-related or AAR-related nucleic acid molecules. Fragments of nucleic acids can also be bound to a physical substrate to comprise what may be considered a macro- or microarray (for example, U.S. Pat. No. 5,837,832; U.S. Pat. No. 5,861,242). Such arrays of nucleic acids may be used to study gene expression or to identify nucleic acid molecules with sufficient identity to the target sequences.
The present invention further provides a nucleic acid array or chip, i.e., a multitude of nucleic acids (e.g., DNA) as molecular probes precisely organized or arrayed on a solid support, which allow for the sequencing of genes, the study of mutations contained therein and/or the analysis of the expression of genes, as such arrays and chips are currently of interest given their very small size and their high capacity in terms of number of analyses.
The function of these nucleic acid arrays/chips is based on molecular probes, mainly oligonucleotides, which are attached to a carrier having a size of generally a few square centimeters or more, as desired. For an analysis, the carrier, such as in a DNA array/chip, is coated with DNA probes (e.g., oligonucleotides) that are arranged at a predetermined location or position on the carrier. A sample containing a target nucleic acid and/or fragments thereof to be analyzed, for example DNA or RNA or cDNA, that has been labeled beforehand, is contacted with the DNA array/chip leading to the formation, through hybridization, of a duplex. After a washing step, analysis of the surface of the chip allows any hybridizations to be located by means of the signals emitted by the labeled target. A hybridization fingerprint results, which, by computer processing, allows retrieval of information such as the expression of genes, the presence of specific fragments in the sample, the determination of sequences and/or the identification of mutations.
In one embodiment of this invention, hybridization between target nucleic acids and nucleic acids of the invention, used in the form of probes and deposited or synthesized in situ on a DNA chip/array, can be determined by means of fluorescence, radioactivity, electronic detection or the like, as are well known in the art.
In another embodiment, the nucleotide sequences of the invention can be used in the form of a DNA array/chip to carry out analyses of the expression of genes involved in the production of AI-2 or in the AAR. This analysis is based on DNA array/chips on which probes, chosen for their specificity to characterize a given gene or nucleotide sequence, are present. The target sequences to be analyzed are labeled before being hybridized onto the chip. After washing, the labeled complexes are detected and quantified, with the hybridizations being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, allows, for example, for differential transcription of RNA derived from the sample.
In yet another embodiment, arrays/chips containing nucleotide sequences of the invention can comprise nucleotide sequences specific for other microorganisms, which allows for serial testing and rapid identification of the presence of a microorganism in a sample.
In a further embodiment, the principle of the DNA array/chip can also be used to produce protein arrays/chips on which the support has been coated with a polypeptide and/or an antibody of this invention, or arrays thereof, in place of the nucleic acid. These protein arrays/chips make it possible, for example, to analyze the biomolecular interactions induced by the affinity capture of targets onto a support coated, e.g., with proteins, by surface plasma resonance (SPR). The polypeptides or antibodies of this invention, capable of specifically binding antibodies or polypeptides derived from the sample to be analyzed, can be used in protein arrays/chips for the detection and/or identification of proteins and/or peptides in a sample.
Thus, the present invention provides a microarray or microchip comprising various nucleic acids of this invention in any combination, including repeats, as well as a microarray comprising various polypeptides of this invention in any combination, including repeats. Also provided is a microarray comprising one or more antibodies that specifically react with various polypeptides of this invention, in any combination, including repeats.
By “nucleic acid molecule” is intended DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A fragment of a nucleic acid molecule encoding an AI-2-related or AAR-related protein may encode a protein fragment that is biologically active, or it may be used as a hybridization probe or PCR primer as described below. A biologically active fragment of a polypeptide disclosed herein can be prepared by isolating a portion of one of the nucleotide sequences of the invention, expressing the encoded portion of the AI-2-related or AAR-related protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the AI-2-related or AAR-related protein. Fragments of nucleic acid molecules encoding AI-2-related or AAR-related proteins comprise at least about 15, 20, 50, 75, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600 nucleotides or up to the total number of nucleotides present in a full-length AI-2-related or AAR-related nucleotide sequence as disclosed herein (for example, 693 for SEQ ID NO:1, 942 for SEQ ID NO:3, 1116 for SEQ ID NO:13, 471 for SEQ ID NO:15, etc.).
Fragments of amino acid sequences include polypeptide fragments suitable for use as immunogens to raise antibodies against polypeptides involved in the production of AI-2 or in the AAR. Fragments include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of an AI-2-related or AAR-related protein, or partial-length protein, of the invention and exhibiting at least one activity of an AI-2-related or AAR-related protein, but which include fewer amino acids than the full-length AI-2-related or AAR-related proteins disclosed herein. In one embodiment, the fragments may include polypeptides that are biologically active. A biologically active portion of a polypeptide is one that exhibits at least one activity (e.g., an activity associated with the production of AI-2 or the AAR) of the full-length protein of the invention. A biologically active portion of an AI-2-related or AAR-related protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500 contiguous amino acids in length, or up to the total number of amino acids present in a full-length AI-2-related or AAR-related protein of the current invention (for example, 231 for SEQ ID NO:2, 314 for SEQ ID NO:4, 372 for SEQ ID NO: 14, 157 for SEQ ID NO:16, etc.). Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native AI-2-related or AAR-related protein. As used here, a fragment comprises at least 5 contiguous amino acids of even SEQ ID NOS:2-22, 34, 36, or 38. The invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400 or greater than 500 amino acids.
AAR-related sequences of the invention comprise SEQ ID NO:19 and 20 which encode a fibronectin binding protein; SEQ ID NO:33, 34, 35 and 36 which encode proteins involved in the production of exopolysaccharides; and SEQ ID NO:37 and 38 which encode a protein involved in D-alanine esterification of lipoteichoic acid and wall teichoic acid. In specific embodiments, a biologically active variant or fragment of SEQ ID NO:19 or 20 will continue to have binding activity to the intestinal mucosa, including binding to fibronectin, mucin or extracellular matrix molecules. Methods to assay for this activity are known. For example, fibronectin binding activity of the variant can be assayed using fluorescent microspheres coated with FN-120, which is a chymotryptic cell-binding fragment of plasma fibronectin. See, for example, Schultz and Armant (1995) J. Biol. Chem. 270(19):11522-31. A biologically active variant or fragment of SEQ ID NO:33, 34, 35, or 36 will continue to be involved in the production of exopolysaccharides. Exopolysaccharide production can be estimated by the phenol/sulphuric acid quantitative method described in Mozi, et al. (2001) J. Appl. Microbiol. 91(1):160-167. A biologically active variant or fragment of SEQ ID NO:37 or 38 will continue to be involved in the D-alanation of lipotechoic acid in bacteria. D-alanine uptake can be measured according to the methods described by O'Brien, et al. (1995) Microbios 83(335):119-137.
Other AAR-related sequences of the invention comprise enzymes involved in the production of AI-2, including SEQ ID NOS:1 and 2, which encode a nucleoside phosphorylase (pfs); SEQ ID NOS:3 and 4, which encode a SAM-dependent methyl transferase (SAM-MT); SEQ ID NOS:13 and 14, which encode a methionine adenosyltransferase (metE); SEQ ID NOS:15 and 16, which encode an S-ribosylhomocysteinase (luxS); and, SEQ ID NOS:21 and 22, which encode a methionine adenosyltransferase (metK). In specific embodiments, a biologically active variant or fragment of SEQ ID NOS:1, 2, 3, 4, 13, 14, 15, 16, 21, or 22 will continue to have the enzymatic activity cited above, respectively. Methods to assay for activity for each of these enzymes are known. For example, metK activity can be measured by quantitating the production of S-adenosyltransferase using high performance liquid chromatography according to the methods described in Posnick and Samson (1999) J. Bacteriol. 181(21):6756-6762. The methyl donor activity of SAM-MT can be assayed according to the methods of Borchardt et al. (1974) J. Med. Chem. 19(9):1104-1110. MTA/SAH nucleoside activity can be measured by following the conversion of 14C-methylthioadenosine to 14C-methylthioribose, as described by Della-Ragione et al. (1995) Biochem. J. 232:335-341 and Cornell et al. (1996) Biochem. J. 317:285-290. MetE activity can be measured according to the assay described by Hondorp and Matthews (2004) PLoS Biol. 2(11):e336. LuxS activity can be measured using the Vibrio harveyi reporter assay as described elsewhere herein.
Variants of the nucleotide and amino acid sequences are encompassed in the present invention. By “variant” is intended a sufficiently identical sequence. Accordingly, the invention encompasses isolated nucleic acid molecules that are sufficiently identical to the nucleotide sequences encoding AI-2-related or AAR-related proteins in even SEQ ID NOS:2-22, 34, 36, or 38 or nucleic acid molecules that hybridize to a nucleic acid molecule of odd SEQ ID NOS:1-21, 33, 35, or 37, or a complement thereof, under stringent conditions. Variants also include variant polypeptides encoded by the nucleotide sequences of the present invention. In addition, polypeptides of the current invention have an amino acid sequence that is sufficiently identical to an amino acid sequence put forth in even SEQ ID NOS:2-22, 34, 36, or 38. By “sufficiently identical” is intended that one amino acid sequence or nucleotide sequence contains a sufficient or minimal number of equivalent or identical amino acid residues or nucleotides as compared to a second amino acid sequence or nucleotide sequence, thus providing a common structural domain and/or indicating a common functional activity. Conservative variants include those nucleotide sequences that differ due to the degeneracy of the genetic code.
In general, amino acid sequences or nucleotide sequences that have at least about 45%, 55%, or 65% identity, at least about 70% or 75% identity, at least about 80%, 85% or 95%, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the amino acid sequences of even SEQ ID NOS:2-22, 34, 36, or 38, or any of the nucleotide sequences of odd SEQ ID NOS:1-21, 33, 35, or 37, respectively, are defined herein as sufficiently identical. Variant proteins encompassed by the present invention are biologically active, that is they retain the desired biological activity of the native protein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
Naturally occurring variants may exist within a population (e.g., the lactic acid bacteria population). Such variants can be identified by using well-known molecular biology techniques, such as the polymerase chain reaction (PCR), and hybridization as described below. Synthetically derived nucleotide sequences, for example, sequences generated by site-directed mutagenesis or PCR-mediated mutagenesis that still encode an AI-2-related or AAR-related protein, are also included as variants. One or more nucleotide or amino acid substitutions, additions, or deletions can be introduced into a nucleotide or amino acid sequence disclosed herein, such that the substitutions, additions, or deletions are introduced into the encoded protein. The additions (insertions) or deletions (truncations) may be made at the N-terminal or C-terminal end of the native protein, or at one or more sites in the native protein. Similarly, a substitution of one or more nucleotides or amino acids may be made at one or more sites in the native protein.
For example, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an “essential” amino acid is required for biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity.
Alternatively, mutations can be made randomly along all or part of the length of the AI-2-related or AAR-related coding sequence, such as by saturation mutagenesis. The mutants can be expressed recombinantly, and screened for those that retain biological activity by assaying for AI-2-related or AAR-related protective activity using standard assay techniques. Methods for mutagenesis and nucleotide sequence alterations are known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. Molecular Biology (MacMillan Publishing Company, New York) and the references sited therein. The mutations made in the DNA encoding the variant should not disrupt the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by comparing the activity of the modified sequence with the activity of the original sequence. For example, the activity of variants and fragments of polypeptides involved in the production of AI-2 or in the AAR can be measured by assaying for the production of AI-2 using methods disclosed elsewhere herein.
Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different AI-2-related or AAR-related protein coding regions can be used to create a new AI-2-related or AAR-related protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the AI-2-related or AAR-related gene of the invention and other known AI-2-related or AAR-related genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Km in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
Variants of an AI-2-related or AAR-related protein of the invention can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an AI-2-related or AAR-related protein. In one embodiment, a variegated library of AI-2-related or AAR-related variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of AI-2-related or AAR-related variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential AI-2-related or AAR-related sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of AI-2-related or AAR-related sequences therein. There are a variety of methods that can be used to produce libraries of potential AI-2-related or AAR-related variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential AI-2-related or AAR-related sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).
In addition, libraries of fragments of an AI-2-related or AAR-related protein coding sequence can be used to generate a variegated population of AI-2-related or AAR-related fragments for screening and subsequent selection of variants of an AI-2-related or AAR-related protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of an AI-2-related or AAR-related coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA which can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, one can derive an expression library that encodes N-terminal and internal fragments of various sizes of the AI-2-related or AAR-related protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of AI-2-related or AAR-related proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify AI-2-related or AAR-related variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
ii. Sequence Identity
The AI-2-related or AAR-related sequences are members of a family of molecules with conserved functional features. By “family” is intended two or more proteins or nucleic acid molecules having sufficient nucleotide sequence or amino acid sequence identity. A family that contains deeply divergent groups may be divided into subfamilies. A clan is a group of families that are thought to have common ancestry. Members of a clan often have a similar tertiary structure. By “sequence identity” is intended the nucleotide or amino acid residues that are the same when aligning two sequences for maximum correspondence over at least one specified comparison window. By “comparison window” is intended a contiguous segment of the two nucleotide sequences or amino acid sequences for optimal alignment, wherein the second sequence may contain additions or deletions (i.e., gaps) as compared to the first sequence. Generally, for nucleic acid alignments, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. For amino acid sequence alignments, the comparison window is at least 6 contiguous amino acids in length, and optionally can be 10, 15, 20, 30, or longer. Those of skill in the art understand that to avoid a high similarity due to inclusion of gaps, a gap penalty is typically introduced and is subtracted from the number of matches.
Family members may be from the same or different species, and can include homologues as well as distinct proteins. Often, members of a family display common functional characteristics. Homologues can be isolated based on their identity to the AI-2-related or AAR-related nucleic acid sequences disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below.
To determine the percent identity of two amino acid or nucleotide sequences, an alignment is performed. Percent identity of the two sequences is a function of the number of identical residues shared by the two sequences in the comparison window (i.e., percent identity=number of identical residues/total number of residues×100). In one embodiment, the sequences are the same length. Methods similar to those mentioned below can be used to determine the percent identity between two sequences. The methods can be used with or without allowing gaps. Alignment may also be performed manually by inspection.
When amino acid sequences differ in conservative substitutions, the percent identity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are known in the art. Typically the conservative substitution is scored as a partial, rather than a full mismatch, thereby increasing the percentage sequence identity.
Mathematical algorithms can be used to determine the percent identity of two sequences. Non-limiting examples of mathematical algorithms are the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; and the search-for-local alignment-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.
Various computer implementations based on these mathematical algorithms have been designed to enable the determination of sequence identity. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. Searches to obtain nucleotide sequences that are homologous to nucleotide sequences of the present invention can be performed with the BLASTN program, score=100, wordlength=12. To obtain amino acid sequences homologous to sequences encoding a protein or polypeptide of the current invention, the BLASTX program may be used, score=50, wordlength=3. Gapped alignments may be obtained by using Gapped BLAST (in BLAST 2.0) as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. To detect distant relationships between molecules, PSI-BLAST can be used. See Altschul et al. (1997) supra. For all of the BLAST programs, the default parameters of the respective programs can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
Another program that can be used to determine percent sequence identity is the ALIGN program (version 2.0), which uses the mathematical algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with this program when comparing amino acid sequences.
In addition to the ALIGN and BLAST programs, the BESTFIT, GAP, FASTA and TFASTA programs are part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Rd., San Diego, Calif., USA), and can be used for performing sequence alignments. The preferred program is GAP version 10, which used the algorithm of Needleman and Wunsch (1970) supra. Unless otherwise stated the sequence identity similarity values provided herein refer to the value obtained using GAP Version 10 with the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
Alignment of a sequence in a database to a queried sequence produced by BLASTN, FASTA, BLASTP or like algorithm is commonly described as a “hit.” Hits to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of a sequence. A hit to a database sequence generally represents an overlap over a fraction of the sequence length of the queried sequence, i.e., a portion or fragment of the queried sequence. However, the overlap can represent the entire length of the queried sequence. The hits in an alignment to a queried sequence produced by BLASTN, FASTA, or BLASTP algorithms to sequences in a database are commonly arranged in order of the degree of similarity and the length of sequence overlap.
Polynucleotide and polypeptide hits aligned by BLASTN, FASTA, or BLASTP algorithms to a queried sequence produce “Expect” values. The Expect value (E value) indicates the number of hits one can “expect” to see over a certain number of contiguous sequences at random when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the GENBANK® or the EMBL database, indicates actual similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the GENBANK® database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score randomly. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 95% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match randomly in the GENBANK® database is 1% or less, using the BLASTN or FASTA algorithm.
According to an embodiment of this invention, “variant” polynucleotides and polypeptides of this invention, comprise sequences producing an E value of about 0.01 or less when compared to the polynucleotide or polypeptide sequences of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described herein. In other embodiments, a variant polynucleotide is a sequence having the same number of, or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described herein. Similarly, a variant polypeptide is a sequence having the same number of, or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described herein.
As noted above, the percentage identity is determined by aligning sequences using one of the BLASTN, FASTA, or BLASTP algorithms, set at the running parameters described herein, and identifying the number of identical nucleic acids or amino acids over the aligned portions; dividing the number of identical nucleic acids or amino acids by the total number of nucleic acids or amino acids of the polynucleotide or polypeptide sequence of the present invention; and then multiplying by 100 to determine the percent identity. For example, a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the GENBANK® database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described herein. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percent identity of the polynucleotide of the present invention to the hit in the GENBANK® library is thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the GENBANK® database is thus not a variant of a polynucleotide of the present invention.
iii. Identification and Isolation of Homologous Sequences
AI-2-related or AAR-related nucleotide sequences identified based on their sequence identity to the AI-2-related or AAR-related nucleotide sequences set forth herein or to fragments and variants thereof are encompassed by the present invention. Methods such as PCR or hybridization can be used to identify sequences from a cDNA or genomic library, for example, which are substantially identical to a sequence of this invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York). Methods for construction of such cDNA and genomic libraries are generally known in the art and are also disclosed in the above reference.
In hybridization techniques, the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may consist of all or part of a known nucleotide sequence disclosed herein. In addition, they may be labeled with a detectable group such as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides, based on the known AI-2-related or AAR-related nucleotide sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known AI-2-related or AAR-related nucleotide sequence or encoded amino acid sequence can additionally be used. The hybridization probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 10, preferably about 20, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of an AI-2-related or AAR-related nucleotide sequence of the invention or a fragment or variant thereof. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among AI-2-related or AAR-related protein sequences. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), herein incorporated by reference.
In one embodiment, the entire nucleotide sequence encoding an AI-2-related or AAR-related protein is used as a probe to identify novel AI-2-related or AAR-related sequences and messenger RNAs. In another embodiment, the probe is a fragment of a nucleotide sequence disclosed herein. In some embodiments, the nucleotide sequence that hybridizes under stringent conditions to the probe can be at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1500 or more nucleotides in length.
Accordingly, in addition to the AI-2-related or AAR-related nucleotide sequences disclosed herein, and fragments and variants thereof, the isolated nucleic acid molecules of the current invention also encompass homologous DNA sequences identified and isolated from other organisms or cells by hybridization with entire or partial sequences obtained from the AI-2-related or AAR-related nucleotide sequences disclosed herein, or variants and fragments thereof.
Substantially identical sequences will hybridize to each other under stringent conditions. By “stringent conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Generally, stringent conditions encompass those conditions for hybridization and washing under which nucleotides having at least about 60%, 65%, 70%, or at least about 75% sequence identity typically remain hybridized to each other. Stringent conditions are known in the art and can be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology (John Wiley & Sons, New York). Hybridization typically occurs for less than about 24 hours, usually about 4 to about 12 hours.
Stringent conditions are sequence dependent and will differ in different circumstances. Full-length or partial nucleic acid sequences may be used to obtain homologues and orthologs encompassed by the present invention. By “orthologs” is intended genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.
When using probes, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
The post-hybridization washes are instrumental in controlling specificity. The two critical factors are ionic strength and temperature of the final wash solution. For the detection of sequences that hybridize to a full-length or approximately full-length target sequence, the temperature under stringent conditions is selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions would encompass temperatures in the range of 1° C. to 20° C. lower than the Tm, depending on the desired degree of stringency as otherwise qualified herein. For DNA-DNA hybrids, the Tm can be determined using the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (logM)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
The ability to detect sequences with varying degrees of homology can be obtained by varying the stringency of the hybridization and/or washing conditions. To target sequences that are 100% identical (homologous probing), stringency conditions must be obtained that do not allow mismatching. By allowing mismatching of nucleotide residues to occur, sequences with a lower degree of similarity can be detected (heterologous probing). For every 1% of mismatching, the Tm is reduced about 1° C.; therefore, hybridization and/or wash conditions can be manipulated to allow hybridization of sequences of a target percentage identity. For example, if sequences with >95% sequence identity are preferred, the Tm can be decreased by 10° C. Two nucleotide sequences could be substantially identical, but fail to hybridize to each other under stringent conditions, if the polypeptides they encode are substantially identical. This situation could arise, for example, if the maximum codon degeneracy of the genetic code is used to create a copy of a nucleic acid.
Exemplary low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. PCR primers are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
iv. Antisense Nucleotide Sequences
The present invention also encompasses antisense nucleic acid molecules, i.e., molecules that are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire AI-2-related or AAR-related coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding an AI-2-related or AAR-related protein. The noncoding regions are the 5′ and 3′ sequences that flank the coding region and are not translated into amino acids. Antisense nucleotide sequences are useful in disrupting the expression of the target gene. Antisense constructions having 70%, 80%, or 85% sequence identity to the corresponding sequence may be used.
Given the coding-strand sequence encoding an AI-2-related or AAR-related protein disclosed herein (e.g., odd SEQ ID NOS:1-21, 33, 35, or 37), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of AI-2-related or AAR-related mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of AI-2-related or AAR-related mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length, or it can be 100, 200 nucleotides, or greater in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation procedures known in the art.
An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330). The invention also encompasses ribozymes, which are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. The invention also encompasses nucleic acid molecules that form triple helical structures. See generally Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807.
In some embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid-phase peptide synthesis protocols as described, for example, in Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670.
In another embodiment, PNAs of an AI-2-related or AAR-related molecule can be modified, e.g., to enhance their stability, specificity, or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) supra; Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.
v. Fusion Proteins
The invention also includes AI-2-related or AAR-related chimeric or fusion proteins. An AI-2-related or AAR-related “chimeric protein” or “fusion protein” comprises an AI-2-related or AAR-related polypeptide operably linked to a non-AI-2-related or AAR-related polypeptide. A “AI-2-related” or “AAR-related polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide that is involved in the production of AI-2 or in the AAR, whereas a “non-AI-2-related” or “non-AAR-related polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially identical to a polypeptide that is involved in the production of AI-2 or in the AAR, and which is derived from the same or a different organism. Within an AI-2-related or AAR-related fusion protein, the AI-2-related or AAR-related polypeptide can correspond to all or a portion of an AI-2-related or AAR-related protein, preferably including at least one biologically active portion of an AI-2-related or AAR-related protein. Within the fusion protein, the term “operably linked” is intended to indicate that the AI-2-related or AAR-related polypeptide and the non-AI-2-related or AAR-related polypeptide are fused in-frame to each other. The non-AI-2-related or AAR-related polypeptide can be fused to the N-terminus or C-terminus of the AI-2-related or AAR-related polypeptide.
Expression of the linked coding sequences results in two linked heterologous amino acid sequences that form the fusion protein. The carrier sequence (the non-AI-2-related or AAR-related polypeptide) can encode a carrier polypeptide that potentiates or increases expression of the fusion protein in the bacterial host. The portion of the fusion protein encoded by the carrier sequence, i.e., the carrier polypeptide, may be a protein fragment, an entire functional moiety, or an entire protein sequence. The carrier region or polypeptide may additionally be designed to be used in purifying the fusion protein, either with antibodies or with affinity purification specific for that carrier polypeptide. Likewise, physical properties of the carrier polypeptide can be exploited to allow selective purification of the fusion protein.
Particular carrier polypeptides of interest include superoxide dismutase (SOD), maltose-binding protein (MBP), glutathione-S-transferase (GST), an N-terminal histidine (His) tag, and the like. This list is not intended to be limiting, as any carrier polypeptide that potentiates expression of the AI-2-related or AAR-related protein as a fusion protein can be used in the methods of the invention.
In one embodiment, the fusion protein is a GST-AI-2-related or AAR-related fusion protein in which the AI-2-related or AAR-related sequences are fused to the C-terminus of the GST sequences. In another embodiment, the fusion protein is an AI-2-related or AAR-related-immunoglobulin fusion protein in which all or part of an AI-2-related or AAR-related protein is fused to sequences derived from a member of the immunoglobulin protein family. The AI-2-related or AAR-related-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-AI-2-related or AAR-related antibodies in a subject, to purify AI-2-related or AAR-related ligands, and in screening assays to identify molecules that inhibit the interaction of an AI-2-related or AAR-related protein with an AI-2-related or AAR-related ligand.
One of skill in the art will recognize that the particular carrier polypeptide is chosen with the purification scheme in mind. For example, His tags, GST, and maltose-binding protein represent carrier polypeptides that have readily available affinity columns to which they can be bound and eluted. Thus, where the carrier polypeptide is an N-terminal His tag such as hexahistidine (His6 tag), the AI-2-related or AAR-related fusion protein can be purified using a matrix comprising a metal-chelating resin, for example, nickel nitrilotriacetic acid (Ni-NTA), nickel iminodiacetic acid (Ni-IDA), and cobalt-containing resin (Co-resin). See, for example, Steinert et al. (1997) QIAGEN News 4:11-15, herein incorporated by reference in its entirety. Where the carrier polypeptide is GST, the AI-2-related or AAR-related fusion protein can be purified using a matrix comprising glutathione-agarose beads (Sigma or Pharmacia Biotech); where the carrier polypeptide is a maltose-binding protein (MBP), the AI-2-related or AAR-related fusion protein can be purified using a matrix comprising an agarose resin derivatized with amylose.
Preferably, a chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences may be ligated together in-frame, or the fusion gene can be synthesized, such as with automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience, New York). Moreover, an AI-2-related or AAR-related-encoding nucleic acid can be cloned into a commercially available expression vector such that it is linked in-frame to an existing fusion moiety.
The fusion protein expression vector is typically designed for ease of removing the carrier polypeptide to allow the AI-2-related or AAR-related protein to retain the native biological activity associated with it. Methods for cleavage of fusion proteins are known in the art. See, for example, Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.). Chemical cleavage of the fusion protein can be accomplished with reagents such as cyanogen bromide, 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine, hydroxylamine, or low pH. Chemical cleavage is often accomplished under denaturing conditions to cleave otherwise insoluble fusion proteins.
Where separation of the AI-2-related or AAR-related polypeptide from the carrier polypeptide is desired and a cleavage site at the junction between these fused polypeptides is not naturally occurring, the fusion construct can be designed to contain a specific protease cleavage site to facilitate enzymatic cleavage and removal of the carrier polypeptide. In this manner, a linker sequence comprising a coding sequence for a peptide that has a cleavage site specific for an enzyme of interest can be fused in-frame between the coding sequence for the carrier polypeptide (for example, MBP, GST, SOD, or an N-terminal His tag) and the coding sequence for the AI-2-related or AAR-related polypeptide. Suitable enzymes having specificity for cleavage sites include, but are not limited to, factor Xa, thrombin, enterokinase, remin, collagenase, and tobacco etch virus (TEV) protease. Cleavage sites for these enzymes are well known in the art. Thus, for example, where factor Xa is to be used to cleave the carrier polypeptide from the AI-2-related or AAR-related polypeptide, the fusion construct can be designed to comprise a linker sequence encoding a factor Xa-sensitive cleavage site, for example, the sequence IEGR (see, for example, Nagai and Thøgersen (1984) Nature 309:810-812, Nagai and Thøgersen (1987) Meth. Enzymol. 153:461-481, and Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309-319, herein incorporated by reference). Where thrombin is to be used to cleave the carrier polypeptide from the AI-2-related or AAR-related polypeptide, the fusion construct can be designed to comprise a linker sequence encoding a thrombin-sensitive cleavage site, for example the sequence LVPRGS or VIAGR (see, for example, Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309-319, and Hong et al. (1997) Chin. Med. Sci. J. 12(3):143-147, respectively, herein incorporated by reference). Cleavage sites for TEV protease are known in the art. See, for example, the cleavage sites described in U.S. Pat. No. 5,532,142, herein incorporated by reference in its entirety. See also the discussion in Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Chapter 16.
vi. Antibodies
An isolated polypeptide of the present invention can be used as an immunogen to generate antibodies that specifically bind AI-2-related or AAR-related proteins, or stimulate production of antibodies against AI-2 related or AAR-related polypeptides in vivo. The full-length AI-2-related or AAR-related protein can be used as an immunogen or, alternatively, antigenic peptide fragments of AI-2-related or AAR-related proteins as described herein can be used. The antigenic peptide of an AI-2-related or AAR-related protein comprises at least 8, preferably 10, 15, 20, or 30 amino acid residues of the amino acid sequence shown in even SEQ ID NOS:2-22, 34, 36 or 38 and encompasses an epitope of an AI-2-related or AAR-related protein such that an antibody raised against the peptide forms a specific immune complex with the AI-2-related or AAR-related protein. Preferred epitopes encompassed by the antigenic peptide are regions of an AI-2-related or AAR-related protein that are located on the surface of the protein, e.g., hydrophilic regions.
vii. Assays
Diagnostic assays to detect expression of the disclosed polypeptides and/or nucleic acid molecules as well as their disclosed activity in a sample are disclosed. An exemplary method for detecting the presence or absence of a disclosed nucleic acid or protein comprising the disclosed polypeptide in a sample involves obtaining a sample from a food/dairy/feed product, starter culture (mother, seed, bulk/set, concentrated, dried, lyophilized, frozen), cultured food/dairy/feed product, dietary supplement, bioprocessing fermentate, or a subject that has ingested a probiotic material, and contacting the sample with a compound or an agent capable of detecting the disclosed polypeptides or nucleic acids (e.g., an mRNA or genomic DNA comprising the disclosed nucleic acid or fragment thereof) such that the presence of the disclosed sequence is detected in the sample. Results obtained with a sample from the food, supplement, culture, product, or subject may be compared to results obtained with a sample from a control culture, product, or subject.
One agent for detecting the mRNA or genomic DNA comprising a disclosed nucleotide sequence is a labeled nucleic acid probe capable of hybridizing to the disclosed nucleotide sequence of the mRNA or genomic DNA. The nucleic acid probe can be, for example, a disclosed nucleic acid molecule, such as the nucleic acid of odd SEQ ID NOS:1-21, 33, 35, 37, or a portion thereof, such as a nucleic acid molecule of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or more nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or genomic DNA comprising the disclosed nucleic acid sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein.
One agent for detecting a protein comprising a disclosed polypeptide sequence is an antibody capable of binding to the disclosed polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(abN)2) can be used. The term “labeled,” with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
The term “sample” is intended to include tissues, cells, and biological fluids present in or isolated from a subject, as well as cells from starter cultures or food products carrying such cultures, or derived from the use of such cultures. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA comprising a disclosed sequence in a sample both in vitro and in vivo. In vitro techniques for detection of mRNA comprising a disclosed sequence include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a protein comprising a disclosed polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of genomic DNA comprising the disclosed nucleotide sequences include Southern hybridizations. Furthermore, in vivo techniques for detection of a protein comprising a disclosed polypeptide include introducing into a subject a labeled antibody against the disclosed polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the sample contains protein molecules from a test subject that has consumed a probiotic material. Alternatively, the sample can contain mRNA or genomic DNA from a starter culture.
The invention also encompasses kits for detecting the presence of disclosed nucleic acids or proteins comprising disclosed polypeptides in a sample. Such kits can be used to determine if a microbe expressing a specific polypeptide of the invention is present in a food product or starter culture, or in a subject that has consumed a probiotic material. For example, the kit can comprise a labeled compound or agent capable of detecting a disclosed polypeptide or mRNA in a sample and means for determining the amount of the disclosed polypeptide in the sample (e.g., an antibody that recognizes the disclosed polypeptide or an oligonucleotide probe that binds to DNA encoding a disclosed polypeptide, e.g., odd SEQ ID NOS:1-21, 33, 35, or 37). Kits can also include instructions detailing the use of such compounds.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a disclosed polypeptide; and, optionally, (2) a second, different antibody that binds to the disclosed polypeptide or the first antibody and is conjugated to a detectable agent. For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, that hybridizes to a disclosed nucleic acid sequence or (2) a pair of primers useful for amplifying a disclosed nucleic acid molecule.
The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container, and all of the various containers are within a single package along with instructions for use.
In one embodiment, the kit comprises multiple probes in an array format, such as those described, for example, in U.S. Pat. Nos. 5,412,087 and 5,545,531, and International Publication No. WO 95/00530, herein incorporated by reference. Probes for use in the array may be synthesized either directly onto the surface of the array, as disclosed in International Publication No. WO 95/00530, or prior to immobilization onto the array surface (Gait, ed. (1984), Oligonucleotide Synthesis a Practical Approach IRL Press Oxford, England). The probes may be immobilized onto the surface using techniques well known to one of skill in the art, such as those described in U.S. Pat. No. 5,412,087. Probes may be a nucleic acid or peptide sequence, preferably purified, or an antibody.
The arrays may be used to screen organisms, samples, or products for differences in their genomic, cDNA, polypeptide, or antibody content, including the presence or absence of specific sequences or proteins, as well as the concentration of those materials. Binding to a capture probe is detected, for example, by signal generated from a label attached to the nucleic acid molecule comprising the disclosed nucleic acid sequence, a polypeptide comprising the disclosed amino acid sequence, or an antibody. The method can include contacting the molecule comprising the disclosed nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having a different plurality of capture probes. The results of each hybridization can be compared to analyze differences in expression between a first and second sample. The first plurality of capture probes can be from a control sample, e.g., a wild type lactic acid bacteria, or control subject, e.g., a food, dietary supplement, starter culture sample or a biological fluid. The second plurality of capture probes can be from an experimental sample, e.g., a mutant type lactic acid bacteria, or subject that has consumed a probiotic material, e.g., a starter culture sample, or a biological fluid.
These assays may be especially useful in microbial selection and quality control procedures where the detection of unwanted materials is essential. The detection of particular nucleotide sequences or polypeptides may also be useful in determining the genetic composition of food, fermentation products, or industrial microbes, or microbes present in the digestive system of animals or humans that have consumed probiotics.
Assays to detect expression of the disclosed polypeptides and/or nucleic acid molecules can also include the detection and/or quantitation of AI-2. Methods for the detection of AI-2 are described elsewhere herein. Assays to measure adherence, for example, in response to adhesion adaptive conditions, can also be measured to evaluate the expression of the polypeptides of the present invention. Such methods are also described elsewhere herein.
III. Recombinant Expression Vectors and Host Cells
The nucleic acid molecules of the present invention may be included in vectors, preferably expression vectors. “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Expression vectors include one or more regulatory sequences and direct the expression of genes to which they are operably linked. By “operably linked” is intended that the nucleotide sequence of interest is linked to the regulatory sequence(s) such that expression of the nucleotide sequence is allowed (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include controllable transcriptional promoters, operators, enhancers, transcriptional terminators, and other expression control elements such as translational control sequences (e.g., Shine-Dalgarno consensus sequence, initiation and termination codons). These regulatory sequences will differ, for example, depending on the host cell being used.
The vectors can be autonomously replicated in a host cell (episomal vectors), or may be integrated into the genome of a host cell, and replicated along with the host genome (non-episomal mammalian vectors). Integrating vectors typically contain at least one sequence homologous to the bacterial chromosome that allows for recombination to occur between homologous DNA in the vector and the bacterial chromosome. Integrating vectors may also comprise bacteriophage or transposon sequences. Episomal vectors, or plasmids are circular double-stranded DNA loops into which additional DNA segments can be ligated. Plasmids capable of stable maintenance in a host are generally the preferred form of expression vectors when using recombinant DNA techniques.
The expression constructs or vectors encompassed in the present invention comprise a nucleic acid construct of the invention in a form suitable for expression of the nucleic acid in a host cell. Expression in prokaryotic host cells is encompassed in the present invention. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., AI-2-related or AAR-related proteins, mutant forms of AI-2-related or AAR-related proteins, fusion proteins, etc.).
Regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain environmental conditions. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (e.g., structural gene) into mRNA. A promoter will have a transcription initiation region, which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, which may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.
An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription. Other examples of positive and negative regulatory elements are well known in the art. Various promoters that can be included in the protein expression system include, but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter. Any suitable promoter can be used to carry out the present invention, including the native promoter or a heterologous promoter. Heterologous promoters may be constitutively active or inducible. A non-limiting example of a heterologous promoter is given in U.S. Pat. No. 6,242,194 to Kullen and Klaenhammer.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature 198:1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publication Nos. 36,776 and 121,775). The beta-lactamase (bla) promoter system (Weissmann, (1981) “The Cloning of Interferon and Other Mistakes,” in Interferon 3 (ed. I. Gresser); bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araB promoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Balbas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed.
In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, the tac (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem. 260:3539-3541) promoters are hybrid trp-lac promoters comprised of both trp promoter and lac operon sequences that are regulated by the lac repressor. The tac promoter has the additional feature of being an inducible regulatory sequence. Thus, for example, expression of a coding sequence operably linked to the tac promoter can be induced in a cell culture by adding isopropyl-1-thio-β-D-galactoside (IPTG). Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publication No. 267,851).
The vector may additionally contain a gene encoding the repressor (or inducer) for that promoter. For example, an inducible vector of the present invention may regulate transcription from the Lac operator (LacO) by expressing the gene encoding the LacI repressor protein. Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp. Alleles of such genes that increase the extent of repression (e.g., lacIq) or that modify the manner of induction (e.g., lambda CI857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) may be employed.
In addition to a functioning promoter sequence, an efficient ribosome-binding site is also useful for the expression of the fusion construct. In prokaryotes, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ end of bacterial 16S rRNA (Steitz et al. (1979) “Genetic Signals and Nucleotide Sequences in Messenger RNA,” in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger, Plenum Press, NY).
AI-2-related or AAR-related proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a protein comprising a signal peptide sequence fragment that provides for secretion of the AI-2-related or AAR-related polypeptides in bacteria (U.S. Pat. No. 4,336,336). The signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids that direct the secretion of the protein from the cell. The protein is either secreted into the growth media (Gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (Gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro, encoded between the signal peptide fragment and the AI-2-related or AAR-related protein.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212). Other prokaryotic signals include, for example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.
Bacteria such as L. acidophilus generally utilize the start codon ATG, which specifies the amino acid methionine (which is modified to N-formylmethionine in prokaryotic organisms). Bacteria also recognize alternative start codons, such as the codons GTG and TTG, which code for valine and leucine, respectively. When they are used as the initiation codon, however, these codons direct the incorporation of methionine rather than of the amino acid they normally encode. Lactobacillus acidophilus NCFM recognizes these alternative start sites and incorporates methionine as the first amino acid.
Typically, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter, flank the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences (of about 50 nucleotides) that are capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
The expression vectors will have a plurality of restriction sites for insertion of the AI-2-related or AAR-related sequence so that it is under transcriptional regulation of the regulatory regions. Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector. Preferred selectable markers include those that confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers may also allow a cell to grow on minimal medium, or in the presence of toxic metabolite and may include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
The regulatory regions may be native (homologous), or may be foreign (heterologous) to the host cell and/or the nucleotide sequence of the invention. The regulatory regions may also be natural or synthetic. Where the region is “foreign” or “heterologous” to the host cell, it is intended that the region is not found in the native cell into which the region is introduced. Where the region is “foreign” or “heterologous” to the AI-2-related or AAR-related nucleotide sequence of the invention, it is intended that the region is not the native or naturally occurring region for the operably linked AI-2-related or AAR-related nucleotide sequence of the invention. For example, the region may be derived from phage. While it may be preferable to express the sequences using heterologous regulatory regions, native regions may be used. Such constructs would be expected in some cases to alter expression levels of AI-2-related or AAR-related proteins in the host cell. Thus, the phenotype of the host cell could be altered.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to AI-2-related or AAR-related mRNA. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen to direct the continuous or inducible expression of the antisense RNA molecule. The antisense expression vector can be in the form of a recombinant plasmid or phagemid in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (1986) Reviews—Trends in Genetics, Vol. 1(1).
Alternatively, some of the above-described components can be put together in transformation vectors. Transformation vectors are typically comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
IV. Microbial or Bacterial Host Cells
Any bacteria of interest can be used in the methods and compositions of the invention. In specific embodiments of the invention, the bacteria employed in the methods is a probiotic bacteria. By “probiotic” is intended a live microorganism that survives passage through the gastrointestinal tract and has a beneficial effect on the subject. As used herein, “probiotic properties” comprises enhanced gut function and stability; improved protection against infectious and non-infectious diseases; immune system modulation; alleviated lactose intolerance; improved digestion and nutrient absorption; reduced blood cholesterol; reduced allergy risk; and reduced risk of urinary tract infections. In some embodiments, an increase in adhesion, stress tolerance, or AI-2 production in a bacterium results in improvement of at least one probiotic property of the bacterium.
In other embodiments of the invention, the bacteria is a lactic acid bacteria. As used herein, “lactic acid bacteria” is intended bacteria from a genus selected from the following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera, Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001) Am. J. Clin. Nutr. 73:365S-373S; Sneath, ed. (1986) Bergey's Manual of Systematic Bacteriology Vol 2, Lippincott, Williams and Wilkins, Hagerstown, Md.).
In still other embodiments, Lactobacillus is used. By “Lactobacillus” is meant any bacteria from the genus Lactobacillus, including but not limited to L. casei, L. rhamnosus, L. johnsonni, L. gasseri, L. acidophilus, L. plantarum, L. fermentum, L. salivarius, L. bulgaricus, and numerous other species outlined by Wood et al. (Holzapfel and Wood, eds. (1995) The Genera of Lactic Acid Bacteria, Vol. 2., Springer, N.Y.) The production of bacteria containing heterologous genes, the preparation of starter cultures of such bacteria, and methods of fermenting substrates, particularly food substrates such as milk, may be carried out in accordance with known techniques, including but not limited to those described in Mayra-Makinen and Bigret (1993) Lactic Acid Bacteria. Salminen and vonWright eds. Marcel Dekker, Inc. New York. 65-96.; Sandine (1996) Dairy Starter Cultures Cogan and Accolas eds. VCH Publishers, New York. 191-206; Gilliland (1985) Bacterial Starter Cultures for Food. CRC Press, Boca Raton, Fla.
By “fermenting” is intended the energy-yielding, metabolic breakdown of organic compounds by microorganisms that generally proceeds under anaerobic conditions and with the evolution of gas.
Nucleic acid molecules or amino acid sequences of the invention may be introduced into host cells by methods known in the art. By “introducing” is intended introduction into prokaryotic cells via conventional transformation or transfection techniques, or by phage-mediated infection. As used herein, the terms “transformation,” “transduction,” “conjugation,” and “protoplast fusion” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and other laboratory manuals.
Bacterial cells used to produce the AI-2-related or AAR-related polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Bacterial strains encompassed by the present invention include those that are biologically pure cultures of a bacterium comprising at least one nucleotide or amino acid sequence of the present invention. These strains may include but are not limited to: Lactobacillus acidophilus, L. gasseri, L. johnsonii, or L. plantarum.
A heterologous expression control sequence or promoter can be operatively associated with a desired nucleotide sequence in accordance with known techniques, such as by targeted insertion or “gene activation” by homologous recombination. See, e.g., U.S. Pat. Nos. 6,391,633 and 6,569,681.
A “subject bacteria or cell” is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, a cell which is descended from a cell so altered and which comprises the alteration, or is a bacteria that has been subjected to adhesion adaptive conditions. A “control” or “control cell” or “control bacteria” provides a reference point for measuring changes in phenotype of the subject bacteria.
A control bacteria may comprise, for example: (a) a wild-type bacteria, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject bacteria; (b) a bacteria of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) bacteria genetically identical to the subject bacteria but which is not exposed to adhesion adaptive conditions or conditions or stimuli that would modulate AI-2 production; or (d) the subject bacteria itself, under conditions in which the gene of interest is not expressed.
V. Methods
i. Modulating the Adhesion Adaptive Response
In one embodiment, the present invention has identified “adhesion adaptive conditions.” As used herein “adhesion adaptive conditions” comprise any physical, chemical, biological, or similar condition that improves the adhesion of bacteria to a substrate. Adhesion can be modulated, for example, by culturing a bacteria to a desired cell density, and then incubating the bacteria under conditions that will enhance the adhesion response of the bacteria. For the purposes of the present invention, “culturing” or a “cell culture” is intended to describe cells that are grown in a synthetic environment. Culture conditions (for example growth media, pH, temperature) vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The cells are typically cultured in favorable conditions to promote cell growth. The cells can be cultured in minimal media (containing the exact nutrients, including any growth factors, needed by the bacteria for growth) or complex media (usually containing the full range of growth factors that may be required for the growth of the bacteria). One can also adjust the physical conditions of a culture medium, such as pH and temperature, to prevent the growth of some organisms while enhancing the growth of others.
In specific embodiments, the bacterial culture conditions comprise culturing anaerobically at 37° C. or 42° C. in Mann-Rogosa-Sharpe (MRS) media. The media can be modified to substitute galactose or other suitable carbohydrates for glucose as the source of sugar in the media. The cells can be harvested at early-log growth phase (defined as having an optical density at 600 nm (OD600) up to 0.4), mid-log growth phase (OD600 around 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9), or late-log growth phase (OD600 greater than 0.9) prior to exposure to adhesive adaption conditions. In specific embodiments, the cells are harvested an OD600 of about 0.6. Bacterial cells are harvested, for example, by sedimentation in a centrifuge or other appropriate device, and resuspended in suitable media.
The harvested bacterial cells can then be preconditioned by incubation under adhesion adaptive conditions (i.e., by incubating the bacterial cells under adhesion adaptive conditions) which results in an increase in adhesion to a target substrate. “Incubating” or “incubation” refers to maintaining a population of bacteria under specific conditions (i.e., adhesion adaptive conditions) in order to promote a particular reaction (for example, an adhesion adaptive response). The adhesion adaptive conditions can include, for example, incubation of bacteria for a time sufficient to increase the adhesion of the bacteria. In some embodiments, the bacteria of the present invention are incubated for at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, or 120 minutes. In other embodiments, the bacteria are incubated under conditions in which the cells have been concentrated to a density of about 1.5×108 to about 1×1010 colony forming units per milliliter (cfu/ml) of diluent (e.g., MRS media), including about 2.0×108, 3.0×108, 4.0×108, 5.0×108, 6.0×108, 7.0×108, 8.0×108, 9.0×108, 1.0×109 or greater cfu/ml. In specific embodiments, the concentration is greater than 1.0×108 cfu/ml. Further embodiments include incubation in a media that has been adjusted to a pH of less than 5, including 4.5, 4.0, 3.5, and 3.0. In specific embodiments, the pH is allowed to naturally fall through a range of about 7.0 to about 4.5. Incubation of bacterial cells under these or other suitable conditions prior to contact of the bacteria to a target substrate (“preconditioning”) can either enhance or diminish, depending on the incubation conditions, the adhesion of the preconditioned bacteria to the target substrate. Any substrate of interest can be employed including, for example, a cell of the gastrointestinal tract or the urogenital tract.
An increase in adhesion comprises any statistically significant increase in adhesion to a target substrate when compared to an appropriate control (for example, bacteria that have not been exposed to adhesion adaptive conditions). In specific embodiments, the increase can comprise, but is not limited to, at least around 90%, 100%, 120%, 130%, 140%, 150%, 200%, 250% or greater. An increase in adhesion can be assayed in vitro by, for example, contacting the preconditioned bacteria to a suitable target (i.e., epithelial or mucosal cells) and counting the number of bacteria that adhere to the target substrate. An increase in adhesion can be assayed in vivo by monitoring host response to exposure to preconditioned bacteria. For example, adhesion of a probiotic bacterium that has been preconditioned under adhesion adaptive conditions can be assessed by monitoring improvement of probiotic properties such as enhanced gut function and stability; improved protection against infectious and non-infectious diseases; immune system modulation; alleviated lactose intolerance; improved digestion and nutrient absorption; reduced blood cholesterol; reduced allergy risk; and reduced risk of urinary tract infections. Additional methods for measuring bacterial adhesion in vivo are described in Leffler, et al. (1995) Methods Enzymol. 253:206-220.
In some embodiments, an increase in adhesion comprises modulation of expression of at least one of SEQ ID NO:19, 20, 33, 34, 35, 36, 37, or 38, or variants and fragments thereof. Further embodiments comprise modulation of expression of 2, 3, 4 or more of these sequences. A modulation in adhesion can be measured by comparing the expression of the polynucleotides of the invention in a bacterium in which the polynucleotides have been introduced to the expression of the same polynucleotide(s) in a control bacterium as defined elsewhere herein. Methods for measuring expression of these sequences are known in the art and are discussed elsewhere herein.
ii. Modulating AI-2 Production
The compositions and methods of the present invention can be used to modulate the production of AI-2 in bacteria. By “modulate,” “alter,” or “modify” is intended the up- or down-regulation of a target biological activity, particularly the up-regulation of activity. Nucleic acid molecules and polypeptides of the invention are useful in modifying the biological activities of lactic acid bacteria, especially lactic acid bacteria that are used to ferment foods with nutritional or health-promoting characteristics. Up- or down-regulation of expression from a polynucleotide of the present invention is encompassed. Up-regulation may be accomplished by providing multiple gene copies, modulating expression by modifying regulatory elements, promoting transcriptional or translational mechanisms, or other means. Down-regulation may be accomplished by using known antisense and gene silencing techniques.
In specific embodiments, the invention provides a bacterium comprising at least one heterologous AI-2 related nucleic acid molecule (i.e., SEQ ID NO: 1, 3, 13, 15, or 21) or biologically active variants or fragments thereof. Further embodiments include a bacterium comprising at least two, at least three, at least four, or at least five AI-2 related nucleic acid molecules as described above. Expression of these heterologous sequences in the bacterium produces an increased level of autoinducer-2 than when compared to an appropriate control bacteria. As used herein, an increase in autoinducer-2 comprises any significant increase in AI-2 over the control. In specific embodiments, the increase can comprise, but is not limited to, at least about 90%, 100%, 120%, 130%, 140%, 150%, 200%, 250% or greater increase in AI-2 production when compared to an appropriate control. Methods to assay for this increase in AI-2 production are discussed in detail elsewhere herein.
The bacteria of the invention having the increase in AI-2 production can display increased adhesion to a target substrate and/or also display improved stress tolerance.
Microbes expressing the polypeptides of the present invention are useful as additives in dairy and fermentation processing. The polynucleotide sequences, encoded polypeptides, and microorganisms expressing them are useful in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk. Microorganisms that express polypeptides of the invention may be probiotic organisms, a lactic acid bacteria, or any other bacterial host of interest.
iii. Improving Stress Tolerance and/or Adhesion
As discussed above, the present invention provides bacteria having improved stress tolerance and/or adhesion activity. As used herein, an improvement in adhesion to a target substrate comprises any significant increase in adhesion to the target substrate including, but not limited to, a 90%, 100%, 120%, 130%, 140%, 150%, 200%, 250% or greater increase in adhesion when compared to an appropriate control. Any substrate of interest can be employed including, for example, a cell of the gastrointestinal tract or the urogenital tract. In specific embodiments, the cell comprises an epithelial cell or a mucosal cell. It if further recognized that the cell can be contacted with the bacteria either in vitro or in vivo.
As used herein, an improved stress-tolerance of a bacteria comprises any significant increase in survival of the bacteria under stress conditions, including, but not limited to, around a 90%, 100%, 120%, 130%, 140%, 150%, 200%, 250% or greater increase in survival when compared to an appropriate control.
A probiotic bacteria of the invention having improved adhesion and/or improved stress tolerance finds use in promoting the heath of a subject. While not intending to be limited by any mechanism of action, probiotic bacteria having these enhanced characteristics upon administration to a subject can block adhesion and compete with pathogens; stimulate host-cell immune defenses; and/or trigger cell-signaling events that “silence” the production of virulence factors from the pathogens. Thus improving the stress tolerance and/or the adhesion activity of a probiotic organism will further aid in reducing the risk of infection of the gut, urogenital tract, and wound sites.
The probiotic bacteria having the increased stress-tolerance and/or increased adhesion can be administered to any subject in need thereof. For example, the subject can comprise a mammal, a human, a domestic animal or an agricultural animal. Administration may be nasal, oral, vaginal or anal. In contexts where mucosal administration is not preferred, the bacterium may be administered by an other suitable means within the capacity of those skilled in the art, i.e., parental routs (i.v., i.p., s.c., i.m.).
It is recognized that the bacteria of the invention having the improved stress-tolerance and/or improved adhesion can further express at least one polynucleotide and/or polypeptide that has therapeutic activity. By “therapeutic activity” is intended that the biological effect when the polypeptide is delivered to a subject is beneficial to that subject. Administration is preferably in a “therapeutically effective amount,” this being sufficient to show benefit to the subject. Such benefit may be at least amelioration of at least one symptom. In a prophylactic context, the amount may be sufficient to reduce the deleterious effect on the individual of a subsequent pathogenic challenge, for instance, by enhancing the immune response. The actual amount administered, rate and time-course of administration, will depend on the aim of the administration, e.g., the biological effect sought in view of the nature and severity of the challenge, and is the subject of routine optimization. Prescription of treatment, including prophylactic vaccination, for example, decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors.
Polynucleotides and/or polypeptides that have therapeutic activity and can be expressed in the probiotic bacteria having improved adhesion activity or improved stress tolerance can include, for example, insulin, growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroid hormone, somatostatin, thyroid-stimulating hormone, vasoactive intestinal polypeptide, a structural group 1 cytokine adopting an antiparallel 4.alpha. helical bundle structure such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-γ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN-α/β, the TNF family of cytokines, e.g., TNFα, TNFβ, CD40, CD27 or FAS ligands, the IL-1 family of cytokines, the fibroblast growth factor family, the platelet-derived growth factors, transforming growth factor .beta. and nerve growth factors, a structural group 3 cytokine comprising short chain α/β molecules, which are produced as large transmembrane precursor molecules which each contain at least one EGF domain in the extracellular region, e.g., the epidermal growth factor family of cytokines, the chemokines characterized by their possession of amino acid sequences grouped around conserved cysteine residues (the C—C or C—X—C chemokine subgroups) or the insulin-related cytokines, a structural group 4 cytokine which exhibits mosaic structures such as the heregulins or neuregulins composed of different domains, e.g., EGF, immunoglobulin-like and kringle domains. Alternatively, the biologically active polypeptide can be a receptor or antagonist for biologically active polypeptides as defined above. In specific embodiments, the polypeptide having therapeutic activity comprises an antigen. For additional therapeutic polypeptide that can be expressed in non-pathogenic bacteria see, for example, U.S. Application 20030202991; Vandenbroucke et al. (2004) Gastroenterology 127:667-8; Huyghebaert et al. (2005) Eur J Pharm Biopharm 59:9-15; Steidler et al. (2000) Science 289:1352-1355; Steidler et al. (2001) The Scientific World 1:215-217; U.S. Application No. 20020019043; each of which are herein incorporated by reference in their entirety. It is recognized that in particular applications, the therapeutic polypeptide may be engineered to be secreted from the probiotic bacteria.
iv. Method of Screening
Method of screening for chemicals or environmental conditions that increase adhesion of a bacterium to a substrate are provided. The method comprises subjecting the bacterium to an environmental condition suspected of increasing adhesion and/or suspected of increasing AI-2 production; and, contacting said bacterium to the substrate. An increase in adhesion of the bacterium subjected to the environmental condition to the substrate compared to adhesion of the same bacterium that has not been subjected to the environmental conditions indicates that the environmental condition is effective in increasing adhesion of the bacterium.
A variety of candidate chemicals or environmental conditions can be employed in this screening method. Such conditions may include, but are not limited to, inducers/suppressors of AI-2 or any gene involved in the production of AI-2, microbiological agents (e.g., prokaryotic and eukaryotic organisms), temperature, concentration, time of incubation in adhesion adaptive conditions, composition of incubation media (e.g., presence, abundance, and/or type of growth factors, carbohydrates, amino acids, salt, minerals, and the like), cell density, or pH. A candidate agent may be a chemical compound, a mixture of chemical compounds, or a biological macromolecule. Such candidate agents can be contained in various agent banks including, for example, compound libraries, peptide libraries, and the like.
The conditions or compounds identified by this process will stimulate bacteria, including both recombinant and non-recombinant wild type bacteria, to produce AI-2, or to promote adhesion or biofilm formation for use in fermentation or probiotic bacteria, or in any other method described herein.
The present invention is explained in greater detail in the examples set forth below.
Gapped BlastP Analysis of Amino Acid Sequences
Sequences of the invention were aligned using the Gapped BlastP alignment method and parameters disclosed herein. Table 1 summarizes the top Blast results for these sequences.
A Gapped BlastP amino acid sequence alignment showed that SEQ ID NO:14 (372 amino acids, ORF LBA1080) has about 55% identity from amino acids 11-371 with a protein from Leuconostoc mesenteroides subsp. mesenteroides that is a methionine synthase II (cobalamin-independent) protein (Accession No. ZP—00064070.1), about 47% identity from amino acids 5-372 with a protein from Lactobacillus gasseri that is a methionine synthase II (cobalamin-independent) protein (Accession No. ZP—00046311.1), about 46% identity from amino acids 7-372 with a hypothetical protein from Chlamydophila pneumoniae (Accession No. NP—224351.1), 44% identity from amino acids 4-372 with a hypothetical protein from Lactobacillus johnsonii (Accession No. NP—965623.1), and 45% identity from amino acids 9-372 with a protein from Oenococcus oeni that is a methionine synthase II (cobalamin-independent) protein (Accession No. ZP—00069898.1).
A Gapped BlastP amino acid sequence alignment showed that SEQ ID NO:16 (157 amino acids ORF LBA1081) has about 87% identity from amino acids 1-157 with a protein from Lactobacillus johnsonii that is an autoinducer-2 production protein LuxS (Accession No. NP—965624.1), about 87% identity from amino acids 1-157 with a protein from Lactobacillus gasseri that is a LuxS protein involved in autoinducer A12 synthesis (Accession No. ZP—00046310.1), about 76% identity from amino acids 4-157 with a protein from Streptococcus bovis that is a LuxS autoinducer 2 synthase (Accession No. dbj|BAD06876.1), 77% identity from amino acids 1-157 with a protein from Lactobacillus plantarum that is an autoinducer production protein (Accession No. NP—784522.1), and 73% identity from amino acids 4-157 with a protein from Streptococcus pyogenes that is an autoinducer-2 production protein (Accession No. NP—269689.1).
A Gapped BlastP amino acid sequence alignment showed that SEQ ID NO:18 (444 amino acids, ORF LBA169) has about 95% identity from amino acids 49-444 with a protein from Lactobacillus acidophilus that is an S-layer protein precursor (Accession No. sp|P358291|SLAP_LACAC), about 67% identity from amino acids 49-443 with a protein from Lactobacillus helveticus that is a surface layer protein (Accession No. emb|CAA62606.1), about 67% identity from amino acids 49-443 with a protein from Lactobacillus helveticus that is a surface layer protein (Accession Nos. emb|CAB46984.1; AJ388558), 66% identity from amino acids 49-443 with a protein from Lactobacillus helveticus that is a surface layer protein (Accession No. emb|CAB46985.1), and 66% identity from amino acids 49-443 with a protein from Lactobacillus helveticus that is a surface layer protein (Accession No. emb|CAB46986.1). A Gapped BlastP amino acid sequence alignment showed that SEQ ID NO:20 (566 amino acids, ORF LBA1148) has about 69% identity from amino acids 4-564 with a hypothetical protein from Lactobacillus johnsonii (Accession No. NP—965038.1), about 66% identity from amino acids 4-564 with a protein from Lactobacillus gasseri that is a predicted RNA-binding protein homologous to a eukaryotic snRNP (Accession No. ZP—00045959.1), about 41% identity from amino acids 4-566 with a protein from Enterococcus faecium that is a predicted RNA-binding protein homologous to a eukaryotic snRNP (Accession No. ZP—00037499.1), about 41% identity from amino acids 4-566 with a protein from Enterococcus faecalis that is homologous to a fibronectin/fibrinogen-binding protein (Accession No. NP—814975.1), and about 41% identity from amino acids 4-557 with a protein from Lactobacillus plantarum that is an adherence protein (Accession No. NP—785358.1).
PFAM Analysis of Amino Acid Sequences
SEQ ID NO:2 (231 amino acids, ORF LBA820) contains a predicted PNP_UDP—1 domain from about amino acids 2 to 222, and is a member of the 5′-methyladenosine phosphorylase (MTA phosphorylase) family (PFAM Accession PF01048).
SEQ ID NO:4 (314 amino acids, ORF LBA931) contains a PrmA domain from about amino acids 6 to 312, and is a member of the Ribosomal protein L11 methyltransferase (PrmA) family (PFAM Accession PF06325).
SEQ ID NO:14 (372 amino acids, ORF LBA1080) contains a predicted Methionine_synt domain from about amino acids 11 to 369, and is a member of the vitamin-B12 independent methionine synthase protein family (PFAM Accession PF01717).
SEQ ID NO:16 (157 amino acids ORF LBA1081) contains a predicted LuxS domain located from about amino acids 2 to 155, and is a member of the LuxS protein family (LuxS) (PFAM Accession PF02664).
SEQ ID NO:20 (566 amino acids, ORF LBA1148) contains a predicted FbpA domain from about amino acids 1 to 425, and is a member of the Fibronectin-binding protein A (FbpA) family (PFAM Accession PF05833).
SEQ ID NO:22 (399 amino acids, ORF LBA1622) contains a predicted S-adenosylmethionine synthetase N-terminal domain from about amino acids 2-102 (PFAM Accession 00438), an S-adenosylmethionine synthetase central domain from about amino acids 124-243 (PFAM Accession 02772), and an S-adenosylmethionine synthetase C-terminal domain from about amino acids 245-384 (PFAM Accession 02773).
Bacterial Strains and Growth Conditions
Lactobacillus strains were cultivated anaerobically at 37° C. or 42° C. in MRS broth (Difco Laboratories Inc., Detroit, Mich.) or, when appropriate, in MRS supplemented with 1.5% agar. Escherichia coli was propagated aerobically in Luria-Bertani (LB, Difco) medium or on LB medium supplemented with 1.5% agar at 37° C. Brain Heart Infusion (BHI, Difco) medium supplemented with 1.5% agar and 150 μg/ml erythromycin (μm) was used for selection of E. coli transformants. Autoinducer Bioassay (AB) media (Bassler et al. (1993) Mol. Microbiol. 9:773-786) was used for the propagation of all Vibrio harveyi strains. When appropriate, chloramphenicol (Cm, 5.0 μg/ml) and Em (5.0 μg/ml or 150 μg/ml) were used for selection. Colony forming units (CFU) per ml were determined with appropriate dilutions using a Whitley Automatic Spiral Plater (Don Whitley Scientific Limited, West Yorkshire, England).
Tissue Culture
The Caco-2 (ATCC HTB-37, Rockville, Md.) cells were only used between the 40th and 50th passage. All reagents used in maintenance of Caco-2 cells were obtained from Gibco (Invitrogen Corp., Carlsbad, Calif.). The cells were routinely grown in 95% air-5% CO2 atmosphere in Minimum Essential Medium (MEM) supplemented with 20% (v/v) inactivated (56° C., 30 min) fetal bovine serum (FBS), 0.10 mM non-essential amino acids and 1.0 mM sodium pyruvate. Monolayers were trypsinized for 10 min, counted using a hemocytometer, and seeded at 1.3×105 cells/well in 2.0 ml of cell culture medium. Cells were grown on 15 mm Thermanox plastic coverslips (Nalge Nunc International, Rochester, N.Y.) in Costar 12-well tissue culture treated plates (Corning Inc., Acton, MA). The medium was replaced every two days and all adherence assays were performed after 14 days of incubation.
Adherence Assay
Adhesion of Lactobacillus strains to Caco-2 cells was examined according to the method described previously (Buck et al. (2005) Appl. Environ. Microbiol. 71:8344-8351). Briefly, mid-log phase bacterial cells (OD600 0.6) were prepared in 10 ml MRS with 3.0 μg/ml Em to maintain selective pressure on integrants. Cells were removed by centrifugation for 10 min at 4000×g, and washed twice with phosphate-buffered saline (PBS). Bacterial pellets were resuspended in 5 ml of fresh MRS prior to adherence. For adhesion adaptive conditions, cell pellets from mid-log phase cultures were resuspended in 1 ml of fresh MRS at ˜1.0×109 cfu/ml and incubated for 1 hr at 37° C. Cells were centrifuged again and resuspended in 5 ml fresh MRS prior to addition to the monolayers. Fifteen-day Caco-2 monolayers were washed twice with PBS and treated with a bacterial suspension at a concentration of 4×108 CFU/ml. Bacteria were incubated on the monolayer for 1.5 hr at 37° C. in a mixture (1:2 v/v) of MRS and cell-line culture medium. Following incubation, the monolayers were washed five times with PBS, fixed in methanol and Gram stained. Adherent bacterial cells were then enumerated microscopically. Duplicate coverslips were counted for each experiment. The final data presented collectively represents at least 3 independent experiments in duplicate. Total counts for each coverslip were used and adhesion was expressed as percent (%) of the control strain NCK1398 (Russell and Klaenhammer (2001) Appl. Environ. Microbiol. 67:4361-4364) which carries an insert in the lacL (β-galactosidase) gene. Using this control, all mutant cultures and the parental control could be prepared with Em to maintain selective pressure on the LuxS− integrant. The adhesion protocol is depicted in
Adhesion Adaptive Response
Ten mL of log phase (OD6000.7) cells were harvested by centrifugation and resuspended in either 1 or 10 mL of fresh Mann-Rogosa Sharpe (MRS) growth medium and incubated for 1 hr at 37 C. Those resuspended in 10 ml are represented as “control conditions” (
Microarray analysis A single 20 mL culture of L. acidophilus was grown in MRS to OD600 of 0.6, divided into two 10 ml aliquots, and harvested by centrifugation for 8 min at 3,150×g. One aliquot was resuspended in 10 ml of fresh MRS, the other resuspended in 1 ml of fresh MRS. Both cultures were then incubated for 1 hr at 37° C. Following incubation, cells were harvested by centrifugation and frozen immediately in a dry ice-ethanol bath. RNA isolation was conducted using TRIzol (Life Technologies, Rockville, Md.) according to the protocol described previously (Azcarate-Peril et al. (2005) Appl. Environ. Microbiol. 71:5794-5804). RNA purity and concentration were determined by electrophoresis on agarose gels and standard spectrophotometer measurements.
Identical amounts (25 μg) of total RNA were aminoallyl-labeled by reverse transcription with random hexamers in the presence of amino-alkyl dUTP (Sigma Chemical Co., St. Louis, Mo.), using Superscript II reverse transcriptase (Clontech Laboratories, Inc., Mountain View, Calif.) at 42° C. overnight, followed by fluorescence-labeling of aminoallylated cDNA with N-hydroxysuccinimide-activated Cy3 or Cy5 esters (Amersham Pharmacia Biotech, Piscataway, N.J.). Labeled cDNA probes were purified using the PCR Purification Kit (Qiagen, Valencia, Calif.). Coupling of the Cy3 and Cy5 dyes to the AA-dUTP labeled cDNA and hybridization of samples to microarrays were performed using standard protocols. Additional information on these protocols can be found on the TIGR website at www.tigr.org/tdb/microarray/protocolsTIGR.shtml. Fluorescence intensities were acquired using a Packard Bioscience ScanArray 4000 microarray scanner (Global Medical Instruments, Inc, Ramsey, Minn.) and processed as TIFF images. Signal intensities were quantified using the GSI Luminomics QuantArray 3.0 software package (PerkinElmer Life and Analytical Sciences, Inc., Boston, Mass.). Two slides (each containing triplicate arrays) were hybridized reciprocally to Cy3- and Cy5-labeled probes per experiment (dye swap). Spots were analyzed by adaptive quantitation (Azcarate-Peril et al., 2005, supra). The local background was subsequently subtracted from the recorded spot intensities. Data was median normalized. The median of the six ratios per gene was recorded. The ratio between the average absolute pixel values for the replicated spots of each gene with and without treatment represented the fold change in gene expression. Confidence intervals and P values on the fold change were also calculated with the use of a two-sample t test. P values of 0.05 or less were considered significant (Table 3).
aORFs shown in bold in table 2 exhibit a similar expression pattern to a previous microarray analysis of L. acidophilus NCFM exposed to MRS acidified to pH 4.5 (3).
Microarray analysis of L. acidophilus NCFM exposed to adhesion adaptive conditions (Table 3) suggests increased production of an interspecies signal, autoinducer-2 (AI-2). The initial (metK) and final (luxS) genes in the pathway for the production of AI-2 from methionine (see
Autoinducer-2 Biosynthetic Pathway
Further analysis of the genome of L. acidophilus NCFM revealed four ORFs (LBA1622, LBA0931, LBA0820, LBA1081) showing homology to each gene in the biosynthetic pathway for the production of AI-2 from methionine (
Table 4 lists other lactobacilli, for which genomic sequence data is available, with the complete pathway for the production of AI-2 from methionine.
The number of predicted ORFs coding for proteins exhibiting similarity to each enzyme in the pathway for synthesis of AI-2 from methionine are listed for L. acidophilus NCFM (Altermann et al. (2005) Proc. Natl. Acad. Sci. USA 102:3906-3912), Lactobacillus gasseri, Lactobacillus johnsonii NCC533 (Pridmore et al. (2004) Proc. Natl. Acad. Sci. USA 101:2512-2517), and Lactobacillus plantarum WCFS1 (Kleerebezum et al. (2003) Proc. Natl. Acad. Sci. USA 100:1990-1995). L. acidophilus NCFM is predicted to harbor two copies of luxS, but one of the ORFs does not contain expression signals such as promoter or ribosomal binding regions.
AI-2 Production
The detection of autoinducer-2 from the supernatant of selected bacterial strains was performed as described previously (DeKeersmaecker and Vanderleyden, 2003, supra) with modifications as follows. The detection of AI-2 produced by lactic acid bacteria can be problematic due to the decreased pH of the spent culture supernatants, and catabolite repression by glucose of the lux operon in the reporter strain V. harveyi BB170 (DeKeersmaecker and Vanderleyden, 2003, supra). Accordingly, all Lactobacillus populations used for detection of autoinducer-2 were grown in modified MRS (mMRS) containing 1% galactose rather than glucose. At specified time points, 4 ml aliquots were collected, OD600 measured, and cell-free supernatants isolated by centrifugation at 4,000×g for 10 min. The pH of the supernatant was neutralized to pH 6.5 with 2N NaOH and filtered through a 0.2 μm membrane. Prepared supernatants were stored at 4° C. until the end of the time course experiments and all samples from each culture were assayed together in separate 96-well microtiter plates. The reporter strain V. harveyi BB170 (Azcarate-Peril, 2005, supra) was grown overnight in autoinducer bioassay (AB) media, washed with and resuspended in fresh AB to OD600 0.5. Ten μl of sterile supernatant was mixed with 90 μl of a 1/1000 dilution of the reporter strain BB 170 in each well of a 96-well plate. Luminescence was measured at 30° C. every 10 min for 6 h in a fluorescent microtiter plater reader (FLOUStar Optima, BMG Technologies, Durham, N.C.). Fold induction was calculated by averaging at least 6 standard time points and dividing the average value obtained from the wild type by the average value obtained from the LuxS− mutant. Each sample was measured in at least 3 independent wells and each data point represents 3 independent samples.
In order to determine when AI-2 was produced during the growth phase of L. acidophilus NCFM, supernatants were harvested at 0, 3, 6, 9, 12, 16, 20, 24, 36, and 48 hrs from triplicate cultures. AI-2 production during the growth of L. acidophilus is shown in
Insertional Inactivation of luxS Gene
Using L. acidophilus NCFM chromosomal DNA as a template, a 471 bp internal fragment of LBA1081 (luxS) was amplified using PCR with primers 1081-IF (5′-GATCA GATCT AAGTT AAGGC ACCTT ACG-3′, SEQ ID NO:23) and 1081-IR (5′-GATCT CTAGA TTTCG AATGG GTCAT CAC-3′, SEQ ID NO:24). The internal fragment was cloned onto the integrative vector pOR128 (Law, et al. (1995) J. Bacteriol. 177:7011-7018) and subsequently transformed by electroporation into L. acidophilus NCFM containing the temperature sensitive helper plasmid pTRK669 (Russell and Klaenhammer, 2001, supra). Steps were then carried out according to Russell and Klaenhammer, 2001, supra for selection of integrants. Successful integration of the plasmid was confirmed by PCR and Southern hybridization analysis of junction fragments using standard protocols.
The LuxS− mutant strain, NCK 1765, was tested for the ability to adhere to Caco-2 cells using bacterial cells from mid-log phase of growth and cells exposed to AAR conditions. When the mid-log phase cells were added directly to the Caco-2 cells from mid-log phase, a 58% decrease in adhesion (Student's t-test, P<0.001) was observed for the LuxS− mutant strain (
L. acidophilus NCFM responds to environmental conditions in a manner that dramatically increases the ability of the organism to adhere to intestinal epithelial cells, in vitro. Transcriptional microarray analysis of a population exposed to these adhesion adaptive conditions suggests that the production of a signaling molecule, AI-2, is increased. Analysis of the genome of L. acidophilus NCFM identified all genes necessary for the production of AI-2 from methionine. Production of AI-2 from L. acidophilus NCFM was confirmed using a V. harveyi reporter strain. Upon inactivation of the final gene in the pathway, luxS, a decrease in adhesion was observed in log phase cells, but no difference was observed in the adhesion adaptive response of the LuxS- mutant strain compared to the control. These data suggest that adhesion of lactobacilli to the intestinal epithelium may involve an intimate interplay of various factors. While AI-2 mediated signaling contributes to the adhesive ability of L. acidophilus NCFM, it is not the exclusive means of communication within the population responsible for the adhesion adaptive response. These data suggest that adhesion of lactobacilli to the intestinal epithelium may involve an intimate interplay of various factors. While AI-2 mediated signaling contributes to the adhesive ability of L. acidophilus NCFM, it is not the exclusive means of communication within the population responsible for the adhesion adaptive response.
Microarray analysis of gene expression affected by LuxS and AI-2 has been performed on a number of different microbial species in order to determine the function of LuxS on cellular processes. The influence of AI-2 on E. coli K-12 grown in the presence and absence of glucose revealed altered gene expression of genes related to the lsr operon, methionine metabolism, and carbon utilization (12, 32). Another study linked LuxS production with genes involved in cellular growth and division using E. coli microarrays (9). Transcriptional microarray analysis of a LuxS− mutant of Porphyromonas gingivalis and its wild type implicated the participation of LuxS with the stress response of that organism (36). In addition to pathogenic bacterial strains, several non-pathogenic strains have demonstrated a phenotype associated with AI-2 production. For example, the ecological performance of Lactobacillus reuteri in the murine gastrointestinal tract was altered in a LuxS− mutant (28). This study utilized a whole genome microarray to identify gene expression influenced by LuxS in L. acidophilus NCFM. Multiple genes involved with the stress response, growth, and metabolism were differentially expressed in a LuxS− mutant strain. Phenotypic analysis of a LuxS− mutant strain examined the influence of a luxS mutation on the heat and bile tolerance of L. acidophilus NCFM.
Bacterial Strains and Growth Conditions.
Lactobacillus strains were cultivated anaerobically at 37° C. or 42° C. in MRS broth (Difco Laboratories Inc., Detroit Mich.) or, when appropriate, in MRS supplemented with 1.5% agar. For detection of AI-2, lactobacilli were grown in modified MRS, following the ingredients for MRS except substituting 1% w/v galactose in place of glucose. E. coli was grown aerobically in Luria-Bertani (LB, Difco) medium or on LB medium supplemented with 1.5% agar at 37° C. Brain Heart Infusion (BHI, Difco) medium supplemented with 1.5% agar and 150 μg/ml erythromycin (Em) was used for selection of E. coli transformants. Autoinducer Bioassay (AB) media (5) was used for the propagation of all Vibrio harveyi strains. When appropriate, chloramphenicol (Cm, 5.0 μg/ml) and Em (5.0 μg/ml or 150 μg/ml) were used for selection. Colony forming units (CFU) per ml were determined with appropriate dilutions in 10% MRS broth using a Whitley Automatic Spiral Plater (Don Whitley Scientific Limited, West Yorkshire, England).
DNA Manipulation Techniques.
Total Lactobacillus genomic DNA was isolated according to the method of Walker and Klaenhammer (31). Standard protocols were used for endonuclease restriction, ligation, DNA modification and transformation (23). Plasmid preparations for the purpose of screening E. coli transformants followed the method of Zhou et al. (37). Large scale plasmid preparations were performed with the QIAprep Spin kit according to the manufacturer's instructions (QIAGEN Inc., Valencia, Calif.). PCRs were carried out according to manufacturer's recommendations using a Taq DNA polymerase PCR system (Roche Applied Science, Indianapolis, Ind.). PCR primers were synthesized by Integrated DNA Technologies (Coralville, Iowa) and, when appropriate, restriction sites were designed into the 5′ end of the primers to facilitate future cloning steps. DNA fragments were extracted from 1.0% agarose gels using the Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, Calif.). Electrocompetent Lactobacillus cells were prepared as described by Walker et al. (30). Southern hybridization of genomic DNA was carried out using standard protocols.
aRestriction enzyme sites used for cloning are underlined.
Marker Free Inactivation of LuxS.
A LuxS− mutant strain of L. acidophilus NCFM was constructed by deletion of 97 bp within luxS (LBA1081) according to the protocols described previously (2, 6). Briefly, a 1,243 bp fragment containing luxS was amplified using primers LuxS-EF and LuxS-ER (Table 6). That fragment was subsequently ligated to pOR128, at the XbaI and BglII sites. Transformants were sleected in E. coli EC1000 and pOR1::LuxSbig was isolated. Inverse PCR (Expand High Fidelity PCR system, Roche Applied Science, Indianapolis, Ind.) was performed on that plasmid using primers LuxS-delF and LuxS-delR (Table 6) each containing EcoRI enzyme recognition sites at the 5′ end. The resulting 2.7 kbp PCR product was cleaned using the Qiagen PCR purification kit and digested for 18 hrs at 37° C. with EcoRI. Following gel purification with the Zymoclean Gel DNA Recovery Kit (Zymogen) the product was ligated to itself for 16 hrs at 16° C. The resulting plasmid, pTRK884, was then transformed into E. coli EC1000, and plasmid construction confirmed with PCR amplification. L. acidophilus NCFM containing the temperature sensitive helper plasmid, pTRK669, was then electrotransformed with pTRK884 and integrants selected according to the method previously described by Russell and Klaenhammer (22). Selected integrants were passed six times in non-selective media, to allow the second cross-over event to occur, and then plated on MRS devoid of antibiotics. Colonies were screened for Em sensitivity and colony PCR conducted using primers LuxS-delNF and LuxS-delNR, flanking the deleted region. PCR amplification of the deletion region and Southern hybridization assays using an internal fragment of luxS as the probe confirmed the deletion via gene replacement in NCK1818.
Until recently, site-specific gene inactivation in lactobacilli has been performed using plasmid integration strategies. Maintenance of plasmid integration requires growth in selective media. However, both plasmid integration and selective growth may influence gene expression results obtained by microarray analysis. As such, a marker-free strategy was used to inactivate luxS (LBA 1081) in L. acidophilus NCFM using a double-crossover homologous recombination approach. The luxS gene of the resulting mutant strain, NCK1818, lacked a 97 bp internal region, and contained an additional EcoRI restriction site. The deletion region was selected to disrupt the reading frame and result in a non-functional gene product. Gene replacement was confirmed by PCR analysis of the deleted region and Southern hybridization using an internal fragment of luxS as the probe. NCK1818 was Em sensitive and did not produce AI-2 as determined by the V. harveyi reporter assay (5). Previously, the pattern of AI-2 expression by L. acidophilus NCFM was determined using a luxS integration mutant as the negative control (8). The same pattern of AI-2 production during the growth of L. acidophilus NCFM was observed using NCK1818 as the negative control. AI-2 detection.
Detection of autoinducer-2 from the supernatant of selected bacterial strains was performed as described previously (8). Briefly, the supernatants from bacterial populations harvested at each specified time point were adjusted to pH 6.5 with 2N NaOH and filtered through a 0.2 μm membrane. The reporter strain Vibrio harveyi BB170 (3) was grown overnight in autoinducer bioassay (AB) media, washed with and resuspended in fresh AB to OD600 0.5. Ten μl of sterile supernatant from the test culture was mixed with 90 μl of a 1/1000 dilution of the reporter strain BB170 in each well of a 96-well plate. Luminescence was measured at 30° C. every 10 min for 6 h in a fluorescent microtiter plate reader (FLOUStar Optima, BMG Technologies, Durham, N.C.). Fold induction was calculated by averaging at least 6 standard time points from the V. harveyi luminescence curve and dividing the average value obtained from the wild type by the average value obtained from the LuxS− mutant. Time points were selected when luminescence was at a constant level before the reporter strain responded to the production of its own AI-2. Each sample was measured in at least 3 independent wells and each data point represents 2 independent experiments (Table 7).
RNA Isolation.
Both L. acidophilus NCFM and NCK1818 were transferred two times from frozen stock cultures in semi-defined media using galactose as the primary carbon source to prevent catabolite repression by glucose of the lux operon in the V. harveyi reporter strain. A 400 ml culture of both strains was inoculated at 1% from a stationary phase (16 hr) culture and allowed to grow at 37° C. At OD600 0.2, a 5 ml sample was obtained for CFU determination and pH measurement. Six 10 ml samples of each strain were then quick cooled on dry ice, and cell pellets harvested at 4° C. by centrifugation for 10 min at 3,150×g. The supernatant from each strain following centrifugation at every time point was retained for AI-2 detection. At the final two sampling points (OD600 0.7 and OD6001.2) 5 ml samples were collected from each culture for plating and pH determination; two 10 ml samples were then harvested from each culture and treated as described above. RNA isolation from cell pellets was conducted as described previously (3) using TRIzol (Invitrogen Life Technologies, Rockville, Md.). RNA purity and concentration were determined by electrophoresis on 1.0% agarose gels and standard spectrophotometer measurements. RNA was isolated from two biological replicates of both strains.
aAll values represent the mean and one standard deviation calculated from combined data obtained from both L. acidophilus NCFM and NCK1818 in three independent experiments.
cDNA Production and Microarray Hybridization.
Gene expression analysis of the LuxS− mutant strain and the wild type strain at each of the three selected sampling points was performed using a whole-genome DNA microarray (3). PCR products of 1,889 predicted ORFs from the L. acidophilus NCFM genome (1) were spotted in triplicate on ULTRA GAPS glass slides (Corning Inc., Acton, Mass.). First strand cDNA synthesis and labeling were performed with the SuperScript Indirect cDNA Labeling System (Invitrogen). Briefly, equal amounts (20 μg) of RNA were aminoallyl-labeled by reverse transcription with random hexamers in the presence of amino-modified nucleotides, using SuperScript III reverse transcriptase at 42° C. for 3 hrs. First-strand cDNA was purified with S.N.A.P columns, precipitated, labeled with N-hydroxysuccinimide-activated Cy3 or Cy5 esters (GE Healthcare Life Sciences, Piscataway, N.J.), and purified again with S.N.A.P columns. The resulting labeled cDNA was hybridized onto microarray slides according to the protocols outlined by TIGR (www.tigr.org/tdb/microarray/protocolsTIGR.shtml). Hybridizations were performed according to a single round-robin pattern, so that all possible direct pair-wise comparisons were conducted in an unbalanced design. A total of fifteen different hybridizations were performed at three different sampling points (OD600 0.2, 0.7, and 1.2) and two different strains (L. acidophilus NCFM and NCK1818).
Microarray Data Analysis.
Fluorescence intensities were acquired using a General Scanning ScanArray 4000 Microarray Scanner (Packard Biochip BioScience, Biochip Technologies LLC, Mass.) and processed as TIFF images. Signal intensities were quantified, including background subtraction, and spot replicates averaged using the QuantArray 3.0 software package (Packard BioScience). The resulting raw intensity data was imported into SAS (SAS Institute, Cary, N.C.), log2 transformed and fit to the normalization mixed model analysis of variance (ANOVA) in order to center the data to the mean intensity. A gene-specific mixed model ANOVA was then performed on the normalized data in which dye, strain, and time were considered fixed effects and the array effect was considered a random effect (34). The resulting difference between least-square estimates for two different treatments is analogous to log2-transformed ratio of gene expression between those two treatments. Differences were calculated for dye effect, strain effect, time effect, and the combined effect of strain and time, strain*time. A t test was performed using these differences and their standard errors, with P<0.05 considered significant. Volcano plots were constructed for each comparison using the estimate and -Log10(P value) with JMP 5.0 (SAS Institute) in order to visualize contrast between treatments and statistical significance of the results.
In order to study the genes differentially expressed in response to LuxS production, a microarray design was developed that examined the differences in expression between the wild type and LuxS− mutant in the early, middle, or late-exponential growth phase. AI-2 production during growth was previously determined for L. acidophilus NCFM (8). From that data, three growth points were selected for RNA isolation: OD600 0.2, before AI-2 has accumulated; OD600 0.7, during the rapid production of AI-2; and, OD600 1.2, when production of AI-2 slows and the level of AI-2 in the supernatant remains elevated but constant. RNA was isolated from both strains at each time point, in duplicate, and AI-2 activity determined using the V. harveyi reporter assay. In total, six points were compared against each other in every possible pair of comparisons (15 hybridizations). Analysis of expression data was performed using a two-stage mixed model ANOVA and least square mean ratios considered significant at a fold change of 1.8 and P<0.05 (Table 9).
Each differentially expressed ORF at the first two time points were grouped according to their COG classification (
A round robin microarray design combined with mixed model analysis was used to compare the effect of strain, time and the combined effect of both strain and time (strain*time) on the gene expression of L. acidophilus NCFM. A strain to strain comparison revealed that, before AI-2 began to appreciably accumulate in the media (OD600 0.2), 84 ORFs were significantly overexpressed in NCFM (Table 8a) while only 13 ORFs were significantly overexpressed in the LuxS mutant (Table 8b). A The most highly induced genes in NCFM compared to the mutant at early-log phase (OD600 0.2) were relA (3.14 fold), putatively related to signal transduction, and IF-2 (3.14 fold), a putative translation initiation factor. At mid-log phase (OD600 0.7), as AI-2 was rapidly accumulating, only 3 ORFs were significantly overexpressed in NCFM (LBA1497, LBA1796, LBA1798) and 4 ORFs (LBA0026, LBA0089, LBA0568, and LBA1596) overexpressed in the LuxS− mutant. Interestingly, the only ORF that was significantly differentially expressed at both of the first two time points was labT (LBA1796), an ABC-transporter previously implicated with bacteriocin export (10). At early-log phase, labT was overexpressed in NCFM 2.53 fold, and at mid-log it was overexpressed 2.10 fold compared to the LuxS− mutant. A response regulator (LBA1798), of a neighboring two component regulatory system, was also overexpressed in NCFM compared to the mutant at mid-log phase. In this regard, the LuxS− strain was analyzed for bacteriocin production and found to elicit the same level of bacteriocin production as the wild type. Additionally, an isogenic labT mutant strain was tested for AI-2 production and found to produce AI-2 at the same level as the wild-type.
aPutative identification by manual annotation for that gene or ORF
bLeast square means ratio of log2 estimates between wild type L. acidophilus NCFM and the LuxS− mutant strain. The cutoff ratio for the fold difference was 1.8 and P < 0.05.
aPutative identification by manual annotation for that gene or ORF
bLeast square means ratio of log2 estimates between wild type L. acidophilus NCFM and the LuxS− mutant strain. The cutoff ratio for the fold difference was 1.8 and P < 0.05.
The trehalose hydrolase, treC (LBA1014), was overexpressed 2.56 fold in NCFM compared to the LuxS− mutant strain at early-log phase. Previous research reported deficient growth on trehalose of a L. acidophilus TreC− mutant (11). Therefore, the maximum growth rate of L. acidophilus NCFM and the LuxS− mutant on mMRS media containing various sugars was measured in order to determine the possible influence of a luxS mutation on sugar utilization (Table 10). Growth was not affected by the luxS mutation when the strains were grown on MRS or mMRS containing glucose or trehalose. However, the LuxS− mutant exhibited a lower maximum growth rate compared to NCFM on both lactose and sucrose.
* Identifies significantly different growth rates as determined by the Student's t test (P < 0.01)
aMedium composition described above
The transcriptional profile of each strain from early to late-log phase was also examined (
Stress Experiments.
Both L. acidophilus NCFM and NCK1818 were grown in MRS broth at 37° C. until the population reached OD600 0.2, 0.7 and 1.2, at which point cells were collected for stress tolerance experiments.
Bile tolerance. At the predetermined sampling points, each culture was diluted and plated in duplicate on MRS agar supplemented with 0.75, 1.0, or 2.0% w/w Oxgall (BD Biosciences, San Jose, Calif.). The plates were incubated anaerobically for 48 hrs and CFU/ml and percent survival were calculated. Each assay was performed in triplicate.
Heat tolerance. Each strain was grown in 250 ml of MRS broth and at each sampling point, 10 ml of cells were harvested from each population by centrifugation at 3,150×g for 10 min at 21° C. Following centrifugation, the supernatant was discarded and cell pellets resuspended in 10 ml fresh MRS, preincubated to 55° C. Samples were taken at 0, 10, 20, 30, and 45 min, diluted, and plated in duplicate. Each assay was performed in triplicate.
Statistical analysis. Data obtained from the above experiments were analyzed using the Student's t test with P<0.05 considered significant.
The involvement of LuxS with various stress responses of L. acidophilus NCFM was tested. Growth on MRS agar supplemented with 2.0% Oxgall (dehydrated fresh bile) significantly decreased the growth of the LuxS− mutant compared to NCFM when bacterial populations were plated at OD600 0.2. When cells were plated from OD600 0.7 populations, a significant decrease in growth was observed in the LuxS− strain on MRS supplemented with 1.0% Oxgall. Cells harvested from the final time point, OD600 1.2, showed no difference in growth on Oxgall (
Additionally, when populations harvested from OD600 0.2 and OD600 0.7 were exposed to 55° C. heat stress, the LuxS− mutant was more sensitive (
Bacteriocin Production
Five μl of both L. acidophilus NCFM and NCK1818 were spotted onto MRS agar and incubated at 37° C. overnight in an anaerobic chamber. The following day, 100 μl of the indicator strain Lactobacillus delbrueckii (NCK235) was added to 10 ml of molten MRS overlay agar (0.75% w/v) and poured evenly onto the surface of the agar plate. After 24 hours of incubation, zones of inhibition indicating antagonistic activity of lactacin B, were evaluated.
Growth Curves
L. acidophilus NCFM and NCK1818 were both transferred three times from frozen stock cultures in MRS and modified MRS (mMRS). The mMRS media used in this study followed the ingredients for commercially available MRS (Becton, Dickinson and Company, Sparks, Md.), replacing dextrose with 1% w/v of either glucose, lactose, trehalose, or sucrose. Growth curves were performed at 37° C. in 96 well plates (Corning) containing 200 μl of each supplemented semi-defined medium or MRS broth. Cultures were allowed to grow for 16 hours and OD600 was measured every 15 min in triplicate wells. Plates were incubated at 37° C., and growth was automatically monitored by determining the changes in A600 as a function of time using a FLUOStar OPTIMA microtiter plate reader (BMG Labtech). The maximum specific growth rate was calculated from the slope of a linear regression line during exponential growth with a correlation coefficient (r2) of 0.99. Each point represented the mean of three independent cultures.
This application is the first to report a transcriptional analysis of the LuxS regulon at different stages of growth. Previous microarray studies of the gene expression modulated by AI-2 have either studied the response of a LuxS− mutant strain to exogenous AI-2 (DeLisa, et al. (2001) Journal of Bacteriology 183:5239-5247) or identified a single growth point and analyzed the transcriptional difference between an insertional mutant and the wild type (Merritt, et al. (2003) Infect Immun 71:1972-9; Wang, et al. (2005) Journal of Bacteriology 187:8350-8360; Yuan, et al. (2005) Infect. Immun. 73:4146-4154). While these approaches can successfully identify genes regulated by AI-2 at single points, the expression profile only represents a snapshot of the LuxS regulon. In order to gain a more complete understanding of gene expression influenced by LuxS, we selected three time points throughout the growth phase of L. acidophilus NCFM for transcriptional analysis. At the first sampling point, OD600 0.2, AI-2 activity detected in the growth media was minimal as the populations were in the early stages of logarithmic growth phase. The second sampling point, during mid-log growth (OD600 0.7), was taken during the rapid accumulation of AI-2 in the media. Cells were harvested at OD600 1.2, late-log phase, for the final sampling point, which was approximately 30 minutes after AI-2 levels reached their peak in the media. These points were chosen to represent the gene expression before, during, and after the production of AI-2 as described above. The LuxS− strain used in this study was a gene deletion mutant and therefore was expected to be free of any pleiotropic effects caused by integrations or addition of selected exogenous components to the media.
The largest number of differentially expressed genes was identified during the early-log phase of growth. A relatively high number of genes were induced in the wild type compared to the LuxS− strain, indicating that AI-2 facilitates the expression of certain genes during the early phases of growth. The majority of these genes were related to transcription, translation, and replication. Three genes (LBA772, LBA773, and LBA775) of the operon encoding the F1F0-ATPase (Kullen and Klaenhammer (1999) Mol. Microbiol. 33:1152-1161) (LBA772-LBA779) were overexpressed in the wild type strain. The trehalose hydrolase (treC, LBA1014) also showed increased expression in the wild type, although no trehalose was present in the media. The absence of AI-2 did not affect the growth of NCFM on trehalose (Table 5), while neither a TreB− (transporter) or TreC− mutant strain were able to grow on trehalose (Duong, et al (2006) Appl. Environ. Microbiol. 72:1218-1225). The differential expression of these putative energy production systems further associates LuxS with growth and metabolism. Similar results obtained by DeLisa et al. (2001, Journal of Bacteriology 183:5239-5247) implicated AI-2 with regulation of cell division, DNA processing, and morphological processes.
In addition to normal growth and metabolic processes, LuxS affected the expression of various cell-surface factors. Two genes (LBA1735 and LBA1736) of a putative exopolysaccharide (EPS) operon were also differentially expressed in the wild type in early-log phase. A putative fibronectin-binding protein, previously shown to participate with adhesion to Caco-2 cells (Buck, et al. (2005) Appl. Environ. Microbiol. 71:8344-8351), was also induced during early-log phase by AI-2. Fibronectin is a component of the human intestinal extracellular matrix (ECM) and could be a target for the adhesion of bacterial cells in the intestinal environment (Kapczynski et al. (2000) Curr. Microbiol. 41:136-41). A LuxS− mutant strain of Lactobacillus reuteri exhibited decreased ecological performance in the murine gastrointestinal tract (Tannock, 2005, supra). Regulating the expression of cell-surface factors early in the growth phase may pre-adapt the bacterium for interaction with a diverse microbial community or ecological performance in the intestinal tract.
Cell-surface associated molecules of intestinal microorganisms are recognized by the host and can be important in both the inflammatory response and maintenance of intestinal homeostatsis (Rakoff-Nahoum, et al. (2004) Cell 118:229-241). One of these bacterial surface molecules, lipoteichoic acid, is bound to the cellular membrane and extends through the cell wall to present itself in the environment of the bacteria. The dltD gene (LBA1923), a member of the dlt operon responsible for proper D-alanation of lipoteichoic acids in lactobacilli, was induced in the presence of LuxS at early-log growth phase. When a Dlt− mutant of Lactobacillus plantarum was exposed to peripheral blood mononuclear cells (PBMCs), the secretion of proinflammatory cytokines TNFα and IL-12 was decreased (Grangette, et al. (2005) Proc. Natl. Acad. Sci. U. S. A. 102:10321-10326). The expression of IL-10 by the PBMCs was increased following exposure to the Dlt mutant, skewing the cytokine profile to an anti-inflammatory response. Regulation by AI-2 of the structure of these lipoteichoic acids could play a role in host-microbe interactions in the intestinal tract.
AI-2 is also thought to participate in the regulation of stress responses in bacteria (Wen, et al. (2004) J. Bacteriol. 186:2682-91; Xavier, et al. (2005) J. Bacteriol. 187:238-248). Accordingly, the expression of multiple genes putatively related with stress responses were influenced by AI-2, including dnaK (LBA1247), grpE (LBA1248), and clpX (LBA847) involved with the removal of misfolded proteins and premature polypeptides produced during heat stress. Analysis of the influence of AI-2 on heat stress survival revealed that when compared to the wild type strain, the LuxS− mutant strain of L. acidophilus NCFM was more sensitive to 55° C. heat stress (
Intestinal bacteria must survive passage through the harsh conditions of the gastrointestinal tract in order to persist in the intestine. One of the hurdles that must be overcome is the exposure to bile in the gastric region. A putative bile salt hydrolase (LBA0892) was induced by LuxS at early-log phase. The LuxS− mutant strain was tested for its tolerance to bile and found to be more sensitive when cultures were harvested from either early or mid-log growth phase. Cultures harvested from late log phase did not exhibit any differences in bile tolerance from the wild type. These results indicate that AI-2 could act to prepare the cell for stressful conditions by positively influencing the expression of genes that have a function with growth and survival in different environmental conditions.
AI-2 was reported to be a quorum sensing factor that accumulates as the environment as cell density increases, although the growth phase in which AI-2 affects gene expression has not been reported. Our results suggest that AI-2 acts on cell populations in the early stages of logarithmic growth, before appreciable accumulation of AI-2. The high number of genes that decreased in expression from early to mid-log growth phase in the LuxS− mutant strain compared to the wild type supports the hypothesis that LuxS positively influences gene expression early in the growth phase. When gene expression was examined throughout the growth phase, LuxS seems to prepare the population for the stresses and interactive conditions naturally encountered during planktonic growth. It is clear that AI-2 impacts the expression of genes related to rapid growth and stress response of L. acidophilus NCFM, as well as possibly influencing host-microbe interactions.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/671,887, filed Apr. 15, 2005, the contents of which are herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60671887 | Apr 2005 | US |
