Patent Application
20130336954
1. Field of the Invention
This invention relates to a nucleic acid encoding a functional module or domain of a particular peptidoglycan hydrolase, i.e., the phage 2638A endolysin, a protein which specifically attacks the peptidoglycan cell wall of untreated Staphylococcus aureus and selected coagulase negative staphylococci (for example: S. chromogenes, S. simulans, S. epidermidis). The invention relates in particular to a full length construct comprising the mutant 180 codon (2638A 1-180 Mut-486) and to truncated constructs encoding a full length amidase domain and the full length SH3b domain, 2638A 139-486 and 2638A 180-486) and to the functional proteins encoded by the constructs.
2. Description of the Relevant Art
The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Staphylococcus aureus has a high negative impact worldwide as a human pathogen and also as a mastitis-causing organism based on its role in infections of dairy cattle mammary glands. S. aureus has a high capacity for resistance development. Resistant S. aureus strains exist to virtually every known antibiotic. Bacteriophage endolysins are proteins encoded by bacteriophage (viruses that infect bacteria) that help nascent phage escape their host by degrading peptidoglycan, the major structural component of bacterial cell walls. Thus, phage and host have co-evolved such that, for those species examined, no endolysin-resistant host strains have been identified (Fischetti, V. A. 2005. Trends Microbiol. 13:491-496), making phage endolysins candidate antimicrobials that are highly refractory to resistance development. To further ensure that our antimicrobials are refractory to resistance development, we have previously created fusion antimicrobials with three active lytic domains, based on the belief that no bacterium can evade three simultaneous, unique, synergistic activities (Donovan et al. 2009. Biotech International 21:6-10).
The bacterial peptidoglycans have a complex structure (sugar backbone of alternating units of N-acetyl glucosamine and N-acetyl muramic acid (NAM) residues, cross-linked by oligopeptide attachments at the NAMs). Endolysins have evolved a modular design to deal with this complexity. One protein can harbor multiple domains, each with a different peptidoglycan digestion activity. Three classes of endolysin domains have been identified thus far: the endopeptidase, glycosidase, and amidase domains (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8: 480-487). Each catalytic domain has been localized to short protein domains (˜100-200 amino acids). Any one of these domains is sufficient to lyse the bacterial target cell.
It has been reported that antibiotic treatment of mastitis is less than 50% effective (Deluyker et al. 2005. J. Vet. Pharmacol. Ther. 22:274-282). S. aureus is also a notorious human pathogen with multi-drug resistant strains plaguing clinics world wide. A new antimicrobial to combat this pathogen would be an excellent addition to the collection of current treatments. There are numerous other bacteriophage endolysins that have been reported to be active against live S. aureus; for example: the phage K endolysin (O'Flaherty et al. 2005. J. Bacteriol. 187:7161-7164; Becker et al. 2008. FEMS Microbiol. Lett. 287:185-191; Becker et al. 2009a. Gene 443:32-41), the lys16 endolysin from the S. aureus phage P68 (Takac et al. 2005. Microbiol. 151:2331-2342), and the lysWMY endolysin from the Staphylococcus warneri M phage (Yokoi et al. 2005. Gene 351:97-108), to name a few.
Antibiotic resistance among pathogens is believed to develop, in part, through the use of broad range antibiotics, which affect not only the target pathogen, but can also select for resistance in other bacteria (e.g. commensals). The use of a highly specific antimicrobial would target fewer species, and thus is less likely to contribute to the broad range resistance development now apparent with commonly used broad range antibiotics. Bacteriophage endolysins are uniquely specific to their host (or closely related species); bacteriophage and bacterial hosts have co-evolved. It is difficult to prove that resistance cannot develop to endolysins, but to date, none has been reported and this fact alone makes this product a candidate for addition to the battery of antimicrobials available to both veterinary medicine and the clinician. If resistant strains are not produced, this would be an important antimicrobial for use and efficacy.
Thus, to manage the upsurge of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents developed specifically for the relevant genera, species or substrains of concern would function as effective tools for controlling economically important diseases and therefore are ideal candidates for therapeutic treatments.
We have discovered that the nucleic acid encoding the endolysin of the staphylococcal phage 2638A comprises a cryptic translational start site in the inter-lytic domain region between the CHAP and amidase domains and that a mutation in codon 180 of the polynucleotide with the cryptic translation start site results in a full length construct comprising the mutant 180 codon (2638A 1-180 Mut-486) and expression of the construct (2638A 1-180 Mut-486) generates a full length 2638A 1-180 Mut-486 endolysin that is lytic for Staphylococcus aureus when exposed externally and that truncated 2638A endolysins comprising the full length amidase domain and the full length SH3 domain confer most of the lytic activity and are dominant to truncated 2638A endolysins comprising either the CHAP domain, or the CHAP domain fused to the SH3b domain.
In accordance with this discovery, it is an object of the invention to provide nucleic acid molecules encoding full length 2638A 1-180 Mut-486 endolysin resulting from mutating codon 180, a cryptic translational start site and nucleic acid molecules encoding the truncated endolysins 2638A 139-486 and 2638A 180-486 comprising the full length amidase domain and the full length SH3b domain.
An added object of the invention is to provide a nucleic acid sequence encoding 2638A endolysin or truncated 2638A endolysin polypeptides according to the invention as an encoding sequence which allows disease resistance to be imparted to the organism. It is well understood that this sequence can also be used in combination with another sequence, or sequences, encoding one or more disease resistant properties.
Another object of the invention is to provide a nucleic acid sequence encoding the uniquely active amidase domain of the 2638A endolysin or truncated 2638A endolysin polypeptides according to the invention as an encoding sequence which allows disease resistance to be imparted to the organism. It is well understood that this sequence can also be used in combination with another sequence, or sequences, encoding one or more disease resistant properties.
It is an object of the invention to provide a nucleic acid sequence encoding 2638A endolysin or truncated 2638A endolysin polypeptides according to the invention as an encoding sequence which can be expressed in the mammary glands of transgenic cattle.
It is a further object of the invention to provide a nucleic acid encoding an antimicrobial fusion protein formed from a nucleic acid encoding a functional module or domain of the 2638A endolysin, a protein which specifically attacks the peptidoglycan cell wall of untreated S. aureus and coagulase negative staphylococci in combination with nucleic acid encoding a functional module(s) or domain(s) of another endolysin(s) having a different hydrolase activity, e.g., glycosidase, amidase and endopeptidase activity.
A still further object of the invention also relates to a chimeric gene (or expression cassette) comprising an encoding sequence as well as heterologous regulatory elements in positions 5′ and 3′ which can function in a host organism, the encoding sequence comprising at least one nucleic acid sequence encoding an antimicrobial 2638A endolysin or a truncated 2638A endolysin.
An additional object of the invention is to provide a host organism into which the 2638A gene, or truncated gene, according to the invention can be introduced so as to produce an endolysin or truncated endolysin.
A further object of the invention is to provide a composition useful for the treatment of disease caused by bacteria for which the full length 2638A endolysin polypeptide is specific.
An added object of the invention is to provide compositions useful for the treatment of disease caused by bacteria for which the full length 2638A endolysin-derived protein having a mutation in position 180 of 2638A endolysin has enhanced, specific antimicrobial activity, given the advantageous antimicrobial activity observed with the full length 2638A endolysin protein and the preserved mutated 2638A endolysin-derived protein together.
A further object of the invention is to provide a composition useful for the treatment of disease caused by bacteria for which the truncated 2638A endolysin-derived proteins are specific.
Also part of this invention is a kit, comprising a composition for treatment of disease caused by the bacteria for which the 2638A endolysin and truncated 2638A endolysin are specific.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
We are interested in identifying staphylococcal endolysins that might serve to impede the escalating development of S. aureus resistant strains. To ensure that our antimicrobials are refractory to resistance development, we have created fusion antimicrobials with three active lytic domains (Donovan et al. 2009, supra), in the belief that no bacterium can evade three simultaneous, unique, synergistic activities. To identify novel domains, we recently collated the SH3b cell wall binding domain containing staphylococcal peptidoglycan hydrolases (Becker et al. 2009a, supra) from public datasets, including many with dual lytic domains. The 486 amino acid 2638A endolysin (Genbank Accession number AAX90995) harbors an N-terminal M23 peptidase domain (retrieved from the Internet: <URL: pfam.sangerac.uk/family?acc=PF01551), a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase; retrieved from the Internet: <URL: (ebi.ac.uk/QuickGO/GTerm?id=GO:0008745), and a C-terminal SH3b—5 (SH3b) cell wall binding domain (retrieved from the Internet: <URL: pfam.sanger.ac.uk/family/PF08239) (See
Phage endolysins are known to be modular in structure (Diaz et al. 1990. Proc. Natl. Acad. Sci. U.S.A. 87:8125-8129; Donovan et al. 2006a. Appl. Environ. Microbiol. 72:2988-2996; Garcia et al. 1990. Gene 86:81-88), and there are numerous examples where single domains are functional without the need for the second lytic domain or the cell wall binding domain (Becker et al. 2009b. FEMS Microbiol. Lett. 294:52-60; Donovan et al. 2006b. Appl. Environ. Microbiol. 72:5108-5112; Donovan et al. 2006c. FEMS Microbiol. Lett. 265:133-139). However, it is still important to demonstrate lysis from without for each endolysin, when considering them as antimicrobials. Toward this end, we isolated the 2638A gene from S. aureus 2854 (HER 1283; University Laval, Quebec, Canada) genomic DNA using PCR cloning, (primers described in Table 2, Example 2) and subcloned this fragment into pET21a (Novagen) E. coli expression vector (construct 2638A 1-486;
We examined, by deletion analysis, the involvement of each of the three domains of 2638A lysin during cell lysis. Deletion variants of the 2638A lysin protein were constructed to isolate each domain on a separate construct so that each domain could be assayed independently. Each deletion variant was His-tagged at the C terminus. Expression of the endolysin constructs was in E. coli (BL21 DE3). All constructs yielded soluble proteins that were purified via (non-Urea) NiNTA nickel column chromatography also previously described (Donovan and Foster-Frey. 2008. FEMS Microbiol. Lett. 287:22-33).
SDS PAGE analysis revealed >90% purity of the resultant purified proteins, except for five of the constructs that extended across the inter-domain region between the peptidase and amidase domains (2638A 1-486; 2638A 1-220::355-486; 2638A 1-411; 2638A 139-411; 2638A 139-486), see Example 3. In these five constructs, there was a second “shadow” band that was consistently co-isolated at high concentration and purity (
All full length constructs and the shadow bands [except for 2638A 1-220::355-486] showed staphylolytic activity (zones of clearing) in the zymogram (
In order to identify the source of the shadow band, it was extracted from the SDS gel from the full length construct 2638A 1-486 sample using standard methods and subjected to Edman degradation N-terminal protein sequencing (M-SCAN, West Chester, Pa.). The last five residues of the resultant amino acid sequence matched perfectly the residues at position 181-184 of the full length 2638A endolysin protein which was consistent with the predicted size of the shadow band from the SDS PAGE (˜37 kD). The N-terminal methionine residue matched the predicted amino acid sequence of a protein expressed from a cryptic translational start site (TTG) at residue 180 thru 486 (36.3 kD), of the published DNA sequence and additional experimental evidence suggested that codon 180 encoded a translational start site.
To test this cryptic translational start site hypothesis, a ninth construct (construct 2638A 1-180Mut-486;
In order to test the activity of the amidase domain together with the SH3b cell wall binding domain construct in the absence of the contaminating shadow band protein, we created a construct via PCR cloning that initiated at codon 180 (2638A 180-486;
The nucleic acid sequences encoding the phage 2638A endolysin-derived proteins: 2638A 1-180 Mut-486, 2638A 139-486, and 2638A 180-486 are identified by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. These sequences include the nucleotides encoding the six histidine tag required for purification. The amino acid sequence of the phage 2638A endolysin-derived protein 2638A 1-180 Mut-486 is identified by SEQ ID NO:4. The truncated endolysin proteins, 2638A 139-486 and 2638A 180-486 are identified by SEQ ID NO:5 and SEQ ID NO:6, respectively. The encoding sequences of the individual modules of the phage 2638A endolysin according to the invention can be assembled by any usual method for constructing and assembling nucleic acid fragments which are well known to those skilled in the art and widely described in the literature and illustrated especially by the use examples of the invention.
Staphylolytic activity was further characterized with two quantitative peptidoglycan hydrolase assays, the turbidity reduction assay and the plate lysis assay, as described previously (Donovan and Foster-Frey, supra).
Another subject of the invention is the use of a nucleic acid sequences encoding the phage 2638A endolysins according to the invention as encoding sequences which allow disease resistance to be imparted to the organism. It is well understood that these sequences can also be used in combination with another sequence, or sequences, encoding one or more disease resistant properties. The present invention therefore also relates to a strategy of generating a nucleic acid sequence encoding a chimeric endolysin according to the invention, this process being defined herein.
The present invention also relates to a chimeric gene (or expression cassette) comprising an encoding sequence as well as heterologous regulatory elements in positions 5′ and 3′ which can function in a host organism, the encoding sequence comprising at least one nucleic acid sequence encoding a phage 2638A endolysin related protein (truncation or fusion) as defined above. By host organism there is to be understood any single-celled or lower or higher non-human multi-celled organism into which a phage 2638A endolysin gene according to the invention can be introduced. The regulatory elements required for expressing the nucleic acid sequence encoding a phage 2638A endolysin are well known to those skilled in the art and depend on the host organism. The means and methods for identifying and choosing the regulatory elements are well known to those skilled in the art and widely described in the literature.
The present invention also relates to a cloning and/or expression vector for transforming a host organism containing at least one of the phage 2638A endolysin genes as defined hereinabove. This vector comprises, in addition, to the above phage 2638A endolysin gene, at least one replication origin. This vector can be constituted by a plasmid, a cosmid, a bacteriophage or a virus which is transformed by introducing the chimeric gene according to the invention. Such transformation vectors according to the host organism to be transformed are well known to those skilled in the art and widely described in the literature.
A further subject of the invention is a process for the transformation of host organisms, by integrating a least one nucleic acid sequence or chimeric gene as defined hereinabove, which transformation may be carried out by any suitable known means which have been widely described in the specialist literature and in particular in the references cited in the present application, more particularly by the vector according to the invention.
According to the present invention, the terms “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, “polynucleotide sequence”, “nucleic acid fragment”, “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded and that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. This will also include a DNA sequence for which the codons encoding the phage 2638A endolysin according to the invention will have been optimized according to the host organism in which it will be expressed, these optimization methods being well known to those skilled in the art.
The term “isolated” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and extrachromosomal DNA and RNA, that normally accompany or interact with it as found in its naturally occurring environment. However, isolated polynucleotides may contain polynucleotide sequences which may have originally existed as extrachromosomal DNA but exist as a nucleotide insertion within the isolated polynucleotide. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
The term “transgene” is understood to describe genetic material which has been or is about to be artificially inserted into the genome of a non-human animal, and particularly into a cell of a living non-human mammal. It is to be understood that as used herein the term “transgenic” includes any cell, cell line, or tissue, the genotype of which has been altered by the presence of a heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
The term “transformation” refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. When the cell is a bacterial cell, the term usually refers to an extrachromosomal, self-replicating vector which harbors a selectable antibiotic resistance. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
The term “construct” refers to a recombinant nucleic acid, generally recombinant DNA, that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences. A “construct” or “chimeric gene construct” refers to a nucleic acid sequence encoding a protein, operably linked to a promoter and/or other regulatory sequences.
The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter) or a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
“Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
The term “cDNA” refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3′ and 5′ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein. “cDNA” refers to a DNA that is complementary to and derived from an mRNA template.
The term “genomic sequence” refers to a sequence having non-contiguous open reading frames, where introns interrupt the protein coding regions. It may further include the 3′ and 5′ untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5′ or 3′ end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.
As used herein, “recombinant” refers to a nucleic acid molecule which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar genetic engineering techniques as described by, for example, Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. or DNA Cloning: A Practical Approach, Vol. I and II (Ed. D. N. Glover), IRL Press, Oxford, 1985. “Recombinant,” as used herein, does not refer to naturally occurring genetic recombinations.
As used herein, the term “chimeric” refers to two or more DNA molecules which are derived from different sources, strains, or species, which do not recombine under natural conditions, or to two or more DNA molecules from the same species, which are linked in a manner that does not occur in the native genome.
As used herein, the terms “encoding”, “coding”, or “encoded” when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to guide translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).
A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
The invention includes functional phage 2638A endolysin polypeptide and functional fragments thereof, as well as mutants and variants having the same biological function or activity. As used herein, the terms “functional fragment”, “mutant” and “variant” refers to a polypeptide which possesses biological function or activity identified through a defined functional assay and associated with a particular biologic, morphologic, or phenotypic alteration in the cell. The term “functional fragments of phage 2638A endolysin” refers to all fragments of phage 2638A endolysin that retain phage 2638A endolysin activity and function to lyse staphylococcal bacteria.
Modifications of the phage 2638A endolysin primary amino acid sequence may result in further mutant or variant proteins having substantially equivalent activity to the phage 2638A endolysin polypeptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may occur by spontaneous changes in amino acid sequences where these changes produce modified polypeptides having substantially equivalent activity to the phage 2638A endolysin polypeptide. Any polypeptides produced by minor modifications of the phage 2638A endolysin primary amino acid sequence are included herein as long as the biological activity of phage 2638A endolysin is present; e.g., having a role in pathways leading to lysis of staphylococcal bacteria.
As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of nucleotides that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. Alterations in a nucleic acid fragment that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a host cell may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (1985. Nucleic Acid Hybridization, Hames and Higgins, Eds., IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. An indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are 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 encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Thus, isolated sequences that encode a phage 2638A endolysin polypeptide and which hybridize under stringent conditions to the phage 2638A endolysin sequences disclosed herein, or to fragments thereof, are encompassed by the present invention.
Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988. CABIOS 4:11-17), the local homology algorithm of Smith et al. (1981. Adv. Appl. Math. 2:482); the homology alignment algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48:443-453); the search-for-similarity-method of Pearson and Lipman (1988. Proc. Natl. Acad. Sci. 85:2444-2448; 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).
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).
A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST. In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification and isolation. In addition, short oligonucleotides of 12 or more nucleotides may be use as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise a particular plant protein. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Thus, such a portion represents a “substantial portion” and can be used to establish “substantial identity”, i.e., sequence identity of at least 80%, compared to the reference sequence. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions at those sequences as defined above.
Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By “fragment” a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby is intended. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence have phage 2638A endolysin-like activity. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes may not encode fragment proteins retaining biological activity.
By “variants” substantially similar sequences are intended. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the phage 2638A endolysin polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR), a technique used for the amplification of specific DNA segments. Generally, variants of a particular nucleotide sequence of the invention will have generally at least about 90%, preferably at least about 95% and more preferably at least about 98% sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein.
By “variant protein” a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended. Variant proteins encompassed by the present invention are biologically active, that is they possess the desired biological activity, that is, phage 2638A endolysin activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native phage 2638A endolysin protein of the invention will have at least about 90%, preferably at least about 95%, and more preferably at least about 98% sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, or even 1 amino acid residue.
The polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Novel proteins having properties of interest may be created by combining elements and fragments of proteins of the present invention, as well as with other proteins. Methods for such manipulations are generally known in the art. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired phage 2638A endolysin activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
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 where the effects of phage 2638A endolysin protein can be observed.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein.
The staphylococcal control compositions of the invention comprise the antimicrobial composition of the invention dissolved or suspended in an aqueous carrier or medium. The composition may further generally comprise an acidulant or admixture, a rheology modifier or admixture, a film-forming agent or admixture, a buffer system, a hydrotrope or admixture, an emollient or admixture, a surfactant or surfactant admixture, a chromophore or colorant, and optional adjuvants. The preferred compositions of this invention comprise ingredients which are generally regarded as safe, and are not of themselves or in admixture incompatible with milk or milk by-products or human and veterinary applications. Likewise, ingredients may be selected for any given composition which are cooperative in their combined effects whether incorporated for antimicrobial efficacy, physical integrity of the formulation or to facilitate healing and health in medical and veterinary applications, including for example in the case of mastitis, healing and health of the teat or other human or animal body part. Generally, the composition comprises a carrier which functions to dilute the active ingredients and facilitates stability and application to the intended surface. The carrier is generally an aqueous medium such as water, or an organic liquid such as an oil, a surfactant, an alcohol, an ester, an ether, or an organic or aqueous mixture of any of these. Water is preferred as a carrier or diluent in compositions of this invention because of its universal availability and unquestionable economic advantages over other liquid diluents.
Avoiding the generalized use of broad range antimicrobials and using highly specific antimicrobials for just the target organisms involved, should help reduce the ever-increasing incidence of antibiotic resistance.
Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
The strains used include numerous S. aureus strains and mastitis isolates, S. chronogenes, S. epidermidis, S. simulans (a gift from M. Paape, USDA, Beltsville, Md.), S. hyicus, S. warneri, and S. xylosus described in Table 1.
S. aureus. Strain
S. aureus. Strain
S. chromogenes
S. epidermidis
S. hyicus
S. simulans
S. warneri
S. xylosus
The staphylococcal phage 2638A lysin gene in pET21a (EMD Biosciences, San Diego, Calif.) was cloned in E. coli DH5a (Invitrogen) and both full length and deletion constructs expressed in BL21 DE3 E. coli cells (Pritchard et al., 2007). Deletion mutants of the phage 2638A lysin protein were constructed with standard molecular techniques (
We isolated the 2638A gene from S. aureus 2854 (HER 1283; University Laval, Quebec, Canada) genomic DNA using PCR cloning, (primers described in Table 2) and subcloned this fragment into pET21a (Novagen) E. coli expression vector (construct 2638A 1-486;
CATATGCTAACTGCT
GTGGTGCT
GAGCACTTC
TGGACGCAA
GAGTTTAATTTCG
GGGGTTATG
In this study, we examined the involvement of each of the three domains of 2638A lysin during cell lysis by deletion analysis. Deletion C-terminal His-tagged variants of the 2638A lysin protein were constructed to isolate each domain on a separate construct so that each domain could be assayed independently. Initially, seven deletion constructs were created via PCR cloning technology in pET21a (conferring a C-terminal 6×His tag) as described previously (Becker et al. 2009b, supra) using the primers in Table 2. All constructs were sequence verified (2638A 1-196; 2638A 1-220; 2638A 1-244; 2638A 1-220::355-486; 2638A 1-411; 2638A 139-411; 2638A 139-486).
Expression of the endolysin constructs was in E. coli (BL21 DE3). All constructs yielded soluble proteins that were purified via (non-Urea) NiNTA nickel column chromatography also previously described (Donovan and Foster-Frey. 2008. FEMS Microbiol. Lett. 287:22-33).
Mid log phase (OD600 nm of 0.4-0.6) E. coli cultures harboring pET21a-derived expression vectors were grown under ampicillin selection, chilled on ice for 30 min, induced with 1 mM IPTG, and incubated with shaking for 18 h at 19° C. Escherichia coli harvested from 100 mL cultures were suspended in 2 mL lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), sonicated on ice for 15×5 s pulses separated by 15 s rests, and centrifuged at 9000 g. for 30 min in a Sorvall HS4 rotor. The cleared supernatant was applied to 1 mL nickel-nitrilotriacetic acid (Ni-NTA) Agarose (nickel matrix) in a slurry and mixed gently for 1 h at 4° C. (Qiagen). The wash and elution buffer profiles were empirically determined for all constructs to be 10 mL of 10 mM imidazole, 20 mL of 20 mM imidazole and eluted into 1.2 mL of 250 mM imidazole in the same phosphate-buffered saline (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). One percent of glycerol was added immediately to the eluate to avoid potential solubility problems that are known to exist for His-tagged proteins (Woestenenk et al., 2004. J. Struc. Func. Genomics 5:217-229). All samples were then either converted to storage buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl with 1% glycerol) via a Zeba desalting column (Pierce) that was previously converted to storage buffer or assayed directly in nickel column elution buffer with 1% glycerol. All samples were 0.22-μm filter sterilized for use in plate lysis assays. Sterilized proteins were stored at 4 or −80° C. until use. Protein concentration determinations were via a BCA Protein kit (Pierce). The purity of each preparation was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The purified constructs and Precision Plus protein standards (Bio-Rad) were analyzed with 15% SDS-PAGE in Tris-Glycine buffer at 150 V for 1.5 h in Criterion Precast gels (Bio-Rad Inc.), according to the manufacturer's instructions. Gels were stained in Coomassie stain for 1 h and then destained for 6-18 h via conventional methods.
SDS PAGE analysis revealed >90% purity of the resultant purified proteins, except for five of the constructs that extended across the inter-domain region between the peptidase and amidase domains (2638A 1-486; 2638A 1-220::355-486; 2638A 1-411; 2638A 139-411; 2638A 139-486,). In these five constructs, there was a second “shadow” band that was consistently co-isolated at high concentration and purity (
In order to identify the source of the shadow band, it was extracted from the SDS gel [from the full length construct 2638A 1-486 sample], using standard methods and subjected to six cycles of Edman degradation N-terminal protein sequencing (M-SCAN, West Chester, Pa.). The amino acid sequence obtained was MKHIYS (SEQ ID NO:22). The last five residues KHIYS (SEQ ID NO:23), matched perfectly the residues at position 181-184 of the full length 2638A endolysin protein (
There are similar levels of expression in the SDS PAGE (
To test the cryptic translational start site hypothesis, a ninth construct with two silent mutations was created where the TTG codon was altered through site-directed mutagenesis to an alternative [CTC] codon that still codes for leucine but did not resemble a translational start site (construct 2638A 1-180Mut-486;
The mutant construct (2638A 1-180Mut-486;
In order to test the activity of the amidase domain together with the SH3b cell wall binding domain construct in the absence of the contaminating shadow band protein, we created a construct via PCR cloning that initiated at codon 180 (2638A 180-486;
The nucleic acid sequences encoding the phage 2638A endolysin-derived proteins: 2638A 1-180 Mut-486, 2638A 139-486, and 2638A 180-486 are identified by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. These sequences include the nucleotides encoding the XhoI cloning site (Leu-Glu) and the six histidine tag required for purification. The amino acid sequence of the phage 2638A endolysin-derived protein 2638A 1-180 Mut-486 is identified by SEQ ID NO:4. The truncated endolysin proteins, 2638A 139-486 and 2638A 180-486 are identified by SEQ ID NO:5 and SEQ ID NO:6, respectively. The recombinant, isolated non-mutated nucleotide sequence (including the nucleotides encoding the XhoI cloning site (Leu-Glu) and the six histidine tag is identified by SEQ ID NO:34. SEQ ID NO:34 encodes the amino acid sequence SEQ ID NO:4.
For Zymogram Analysis: Zymogram gels were loaded (5 μg) and electrophoresed identically. Zymogram contained S. aureus strain Newman, were rinsed in water to remove SDS and soaked in 50 mM Phosphate, 150 mM NaCl, pH 7.5 for 2.5 hours. Lane and predicted molecular weights of each construct: Lane M, Kaleidoscope Molecular Weight Markers (Biorad); Lane 1, 2638A 1-486, 56.6 kD; Lane 2, 2638A 1-196, 23.4 kD; Lane 3, 2638A 1-220, 25.9 kD; Lane 4, 2638A 1-244, 28.6 kD; Lane 5, 2638A 1-220::355-486, 41.25 kD; Lane 6, 2638A 1-411, 48 kD; Lane 7, 2638A 139-411, 32.4 kD; Lane 8, 2638A 139-486, 40.9 kD; Lane 9, 2638A 180-486, 36.3 kD; Lane 10, 2638A 1-180Mut-486, 56.6 kD.
All full length constructs and the shadow bands [except for 2638A 1-220::355-486] showed staphylolytic activity (zones of clearing) in the zymogram (
It is apparent from the zymogram that both the M23 peptidase domain and the amidase domain are enzymatically active on SDS treated S. aureus cells.
The turbidity assay measures the drop in optical density (OD) resulting from lysis of the target bacteria with the phage endolysin-derived protein. Mid log phase (OD600 nm of 0.4-0.6) target cells were grown in Brain Heart Infusion (Becton Dickenson, Franklin Lakes, N.J.) and concentrated in lysing buffer A (LBA; 50 mM ammonium acetate, 10 mM CaCl2, 1 mM DTT at pH 6.2) to an OD600 nm of ˜2.0. The turbidity reduction assays were performed with identical molar amounts of proteins and S. aureus strain Newman with the addition of the bivalent metal cations Mn2+, Mg2+, and Ca2+ at a concentration of 1 mM. The turbidity reduction assay contained 0.5 μM protein (5.7 μg of full length repaired construct: 2638A 1-180Mut-486 in 200 μl assay) unless otherwise noted. Lane 1, 2638A 1-486 (5.7 μg total protein in 200 μl assay); Lane 2, 2638A 1-196; Lane 3, 2638A 1-220; Lane 4, 2638A 1-244; Lane 5, 2638A 1-220::355-486; Lane 6, 2638A 1-411; Lane 7, 2638A 139-411; Lane 8, 2638A 139-486; Lane 9, 2638A 180-486; Lane 10, 2638A 1-180Mut-486. Mixing reactions between the repaired full length construct 2638A 1-180Mut-486 (R) and the engineered amidase-SH3b construct 2638A 180-486 (A) were performed in the following ratios Lane 11, R:A::1:1, 1 μM; Lane 12, R:A::1:1, 0.5 μM; Lane 13, R:A::1:3, 0.5 μM; and Lane 14, R:A::3:1, 0.5 μM. Optical Density measurements are taken at regular intervals. If the lysin can digest the cell wall, lysis will occur with a subsequent reduction in OD. Changes in the OD600 nm in the control sample (cells alone) were subtracted from samples containing both cells and lysin, before calculating the specific activity. Specific Activity=ΔOD600 nm/μM/min.
The turbidity reduction assay results indicate that the parent full length construct (2638A 1-486), including its shadow band, shows the highest activity in the turbidity reduction assay (
The exact ratio of shadow band: full length construct is unknown as they are produced and purified simultaneously in the nickel column purified preparation. Protein sequence analysis described above indicates that the shadow band produced by the full length construct (2638A 1-486) is the same protein as produced by construct 2638A 180-486. It was reasoned that the protein mixture might be the source of the enhanced activity. To test this hypothesis, a series of mixing experiments were performed where defined molar amounts of both the repaired full length construct (2638A 1-180Mut-486) and the 2638A 180-486 amidase construct were added in the turbidity reduction assay in an effort to mimic the ratio of full length to shadow band produced by the parent construct (2638A 1-486). Although it is impossible to know the exact concentration of the full length and shadow band in the 2638A 1-486 construct, 0.5 μM of the full length repaired construct is 11 μg of protein. Thus 11 μg of the full length+shadow band was used in the turbidity reduction assay for comparison. Molar Ratios of 1:1, 1:3 and 3:1 (2638A 1-180Mut-486 repaired: 2638A 180-486 amidase), performed at room temperature (
There was weak turbidity reduction activity from the 2638A 1-486 parental construct on methicillin resistant S. aureus (MRSA) strain (CSA #175, SRCAMB collection) and no activity on S. epidermidis (ATCC 14990) (data not shown).
The presence and use of the codon 180 TTG cryptic translational start site in a heterologous E. coli expression system begs the question of whether or not this codon 180 translational start site is functional in S. aureus. Our results do not address this question specifically, but one study suggests that TTG translational start codons are used in 8% of the S. aureus genes examined, a much higher frequency than the 2% of E. coli genes cited in the same work (Starmer et al., supra). A search for S. aureus SD sequences has identified several: AGAGAG, AGAAAG (Strommenger et al. 2004. Eur. J. Clin. Microbiol. Infect. Dis. 23:15-19), GGAGGG (East and Dyke. 1989. J. Gen. Microbiol. 135:1001-1015), AAAGGAG (Jones and Khan. 1986. J. Bacteriol. 166:29-33) and AAAGGAAGGAATTA (SEQ ID NO:24; Cuny and Witte. 1996. J. Clin. Microbiol. 34: 1502-1505). A cursory comparison of these published sequences to the DNA sequences shown in
Turbidity reduction assays suggest that when both the full length and the shadow band protein are putatively produced from the same transcript, there is a heightened endolysin activity derived. If there is a selective advantage to this heightened activity, this might explain why this sequence has been maintained over time.
Thus, the 2638A endolysin is a potent antimicrobial with a uniquely active amidase domain that will be a good addition to future antimicrobial constructs. The 2638A endolysin constructs can be used in novel environments to determine if the unique plate lysis phenotype is predictive of novel environments where this endolysin will find special application.
Purified proteins for each construct were diluted in sterile nickel column elution buffer, and 6 μL of lysostaphin (11 μg) or the constructs (0.2 nmoles or ˜11 μg for the repaired construct 2638A 1-180Mut-486) was spotted onto a freshly spread lawn of S. aureus strain NRS119 (SA LinR #12; linezolid resistant) growing cells that had air dried for 30 min on tryptic soy agar (TSA) plates. L=1 μg Lysostaphin (Sigma); Spot 1=11 μg; all other constructs are 0.2 nmoles (˜11 μg for the repaired construct 2638A 1-180Mut-486) spotted in 10. The spotted plates were air dried for 10 min in a laminar flow hood and incubated overnight in a 37° C. environment. Scoring of the cleared spots occurred within 20 h of plating the cells.
The Plate Lysis assay results (
The finding that the 2638A amidase domain is highly active and the M23 peptidase domain appears nearly inactive is unexpected, and in direct opposition to the results of studies with similar proteins, e.g. the staphylococcal LysK (phage K endolysin) and phage phi11 endolysin. Despite a virtually identical protein organization in all three proteins, peptidase-amidase-SH3b, the LysK amidase domain was virtually inactive in constructs where it was isolated, although it was shown to be active in the context of the whole protein (Becker et al. 2009b, supra). Similarly, the phi11 endolysin amidase domain was virtually inactive when isolated in a deletion construct (Sass and Bierbaum. 2007. Appl. Environ. Microbiol. 73:347-352). In contrast, the cysteine, histidine-dependent amido-hydrolases/peptidases (CHAP) endopeptidase (Bateman and Rawlings. 2003. Trends Biochem. Sci. 28:234-237; Rigden et al. 2003. Trends Biochem. Sci. 28:230-234) domain isolating constructs from both the phi11 endolysin (Donovan et al. 2006c., supra; Saas and Bierbaum, supra) and LysK (Becker et al. 2009b, supra; Horgan et al. 2009. Appl. Environ. Microbiol. 75:872-874) demonstrate strong lytic activity. Despite readily observed zones of clearing in the zymogram (
Expression and purification of recombinant, C-terminally 6×His-tagged phage endolysins were performed essentially as previously described (Donovan and Foster-Frey, supra), with the following modifications: Induced E. coli cultures were harvested, resuspended in 10 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 30% glycerol, pH 8.0) per 1 L culture, and sonicated on ice for 5 min (1 s pulses separated by 1 s rests). After removal of debris by centrifugation (9000×g for 30 min), 6×His-tagged proteins were purified from the cleared supernatant by immobilized metal ion affinity chromatography, using nickel-NTA Superflow resin (QIAGEN, Valencia, Calif.). Purification columns were washed with 25 column volumes (CV) of lysis buffer supplemented with 0.1% Triton X-114 for removal of endotoxins (Reichelt et al. 2006. Prot. Express. Purif. 46:483-488), 40 CV of lysis buffer, and 15 CV of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 30% glycerol, pH 8.0). Target proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 30% glycerol, pH 8.0) in 500 μl fractions. Fractions with high protein concentrations were combined and dialyzed against Dialysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 7.5). Protein concentrations were measured spectrophotometrically using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, Del.), and purity was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Endotoxin concentrations were determined using a Limulus Amoebocyte Lysate (LAL) assay (Lonza, Walkersville, Md.). Endolysin activity was assayed using the plate lysis method essentially as described earlier (Becker et al. 2009b, supra).
Four to six week old female Balb/c mice (weight range 22 g to 24 g, Harlan Laboratories) were used in biosafety level 2 facilities in accordance with IACUC regulations. Briefly, methicillin-resistant Staphylococcus aureus (MRSA) strain NRS382, acquired from NARSA (Network on Antimicrobial Resistance in Staphylococcus aureus, Chantilly, Va.), was grown at 37° C. overnight in Brain Heart Infusion (BHI) medium (Becton, Dickinson and Company, Sparks, Md.). The culture was then diluted 1:100 and grown to mid-log phase (OD600 nm=0.3-0.4), centrifuged and resuspended in BHI supplemented with 5% mucin (Sigma-Aldrich St Louis, Mo., USA) for the mouse experiment. Mucin functions as immunosuppressant and allows reduction of the bacterial inoculum concentration required to achieve an LD90 after 48 hours. Approximately 4×107 CFU bacteria in suspension (in a volume of 0.2 ml) were injected intraperitoneally (I.P.). Actual inoculum titers were derived from plating serial dilutions of each inoculum on BHI agar plates.
To determine the in vivo efficacy of 2638A endolysin, 30 minutes post infection, infected mice were divided into several groups (ten mice in each group) and were I.P. injected with 2638A (SEQ ID NO:4) in Dialysis Buffer (200 μg/mouse), or phosphate-buffered saline (PBS) or Dialysis Buffer as controls (0.2 ml/mouse). The antibiotics Vancomycin and Oxacillin (Sigma-Aldrich, St Louis, Mo., USA), prepared in distilled H2O, were used as additional controls. Antibiotics were administrated subcutaneously (Vancomycin: 375 μg/mouse; Oxacillin: 1250 μg/mouse) 30 minutes post infection (
The survival rate for each experimental group was monitored every 4 hours up to 48 hours post infection. The data were statistically analyzed by Kaplan Meier Survival curves. In addition, a Septicemia Score Index described by Biswas et al. (2002. Infect. Immun. 70:204-210) was used to evaluate the health condition of MRSA-infected mice in intervals of 4 hours for up to 48 hours (Table 4).
Endolysin 2638A protects mice from MRSA-induced bacteremia. The percentage of mice surviving after intraperitoneal injection of MRSA was monitored for 48 hours. For mice treated with PBS, Dialysis Buffer, or Oxacillin, the survival rate dropped from 100% to less than 40% after 16 to 20 hours, and reached approximately 20% at the end of the experiment (
In order to detect bacteria in the bloodstream, mice surviving until the end of the experiment (48 hours post infection) were euthanized, 100 μl blood samples were taken, mixed with 900 μl of PBS, and then serially diluted and plated on BHI agar plates.
Septicemia scores of animals treated with PBS, Dialysis Buffer, or Oxacillin continuously increased after the treatment and reached an average of approximately 4 (corresponding to a moribund disease state; see Table 4) at 20 hours post infection, which was maintained until the end of the experiment (
All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.
