Patent Application
20220017573
Aspects herein relate generally to peptides and peptide-responsive regulators. More particularly, some aspects herein relate to peptides, culture media, compositions, methods and uses for inducing bacteriocin production in a microbial organism. Some other aspects herein relate to a method for identifying, selecting and/or optimizing peptide ligands of peptide-responsive regulators.
Populations of microbial organisms are involved in maintaining the health and metabolic functions of multicellular organisms, for example as the microbiota associated with the gut and skin of humans, or the roots of the plants. In addition, populations of microbial organisms are used for various industrial processes. Accordingly, tuning populations of microbial organisms, for example to reduce or eliminate or neutralize undesired microbial organisms, can be useful for maintaining robustness and consistency of industrial processes and maintaining the health of tissues that comprise microbial organisms. For example, WO 2015/024855 describes systems, methods and microbial cells for the controlled growth of microorganisms.
Extensive and widespread use of antimicrobial drugs to reduce or eliminate or neutralize undesired microbial organisms has led to the emergence of resistant strains of microbial organisms. These microbial organisms are no longer susceptible to the currently available antimicrobial drugs. Bacteriocins are proteinaceous or peptidic toxins produced by microbial organisms, typically to inhibit the growth of similar or closely related strain(s). Bacteriocins are able to overcome at least some of the drawbacks associated with antimicrobials, as they are still active against antimicrobial-resistant microbial organisms.
It is well known that regulation of bacteriocin production is tightly coupled to regulation of competence. Competence has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a physiological state enabling bacteria to bind and take up high-molecular-weight exogenous DNA (transformation). Concomitant induction of competence upon inducing bacteriocin production may be disadvantageous, as it places a fitness burden on the microbial population, and increases the likelihood of unwanted genetic changes occurring in the microbial organisms in the population. For example, competence may increase the spread of antibiotic resistance genes and/or bacteriocin immunity genes.
Accordingly, to expand the currently available arsenal of compounds that can be used to tune populations of microbial organisms, there is a need for compounds which can activate bacteriocin production in microbial cells, preferably without concomitantly inducing competence to avoid the disadvantages thereof as indicated above.
In a first aspect, there is provided a peptide or peptidomimetic with a length of at least 6 residues comprising, consisting essentially of, or consisting of the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 1), wherein:
Preferably, the peptide or peptidomimetic is able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence.
In a preferred embodiment, the peptide or peptidomimetic has a maximum length of 30 amino acids. In another preferred embodiment, the peptide or peptidomimetic has a maximum length of 20 amino acids. In another preferred embodiment, the peptide or peptidomimetic has a maximum length of 10 amino acids.
In a second aspect, there is provided a polypeptide able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising a peptide or peptidomimetic as described in the first aspect, wherein the peptide or peptidomimetic can be released from the polypeptide by natural, chemical or biological peptide hydrolysis.
In a third aspect, there is provided a culture medium comprising a peptide or peptidomimetic as described in the first aspect. Also provided is a culture medium comprising a peptide or peptidomimetic according to to the first aspect and/or a polypeptide according to the second aspect. The culture medium may further comprise a signaling molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin.
In a fourth aspect, there is provided a composition for inducing bacteriocin production in a microbial organism, comprising a peptide or peptidomimetic as described in the first aspect and a solvent. Also provided is a composition for inducing bacteriocin production in a microbial organism, comprising a peptide or peptidomimetic according to the first aspect and/or polypeptide according to the second aspect. The composition may further comprise a signaling molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin.
In a fifth aspect, there is provided a microbial organism able to produce and/or secrete a peptide or peptidomimetic according to the first aspect and/or a polypeptide according to the second aspect.
In a sixth aspect, there is provided a method for inducing bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising administering a peptide or peptidomimetic according to the first aspect to the microbial organism, and/or culturing the microbial organism in a culture medium according to the third aspect, and/or administering a composition according to the fourth aspect to the microbial organism. The bacteriocin production in the microbial organism can neutralize a second, undesired microbial organism. According to some embodiments, the microbial organism is a Gram-positive bacterium, for example a lactic acid bacterium, such as a Streptococcus species, such as Streptococcus salivarius.
Also provided is a method for inducing bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising administering a peptide or peptidomimetic according to the first apect and/or a polypeptide according to the second aspect to the microbial organism, and/or culturing the microbial organism in a culture medium according to the third aspect, and/or administering a composition according to the fourth aspect to the microbial organism. Also provided is a method for inducing bacteriocin production in a first microbial organism, preferably without concomitantly inducing competence, comprising administering to the first microbial organism a second microbial organism according to the fifth aspect and/or co-culturing the first microbial organism with the second microbial organism according to the fifth aspect. Preferably, the bacteriocin-producing microbial organism belongs to the microbiota. Preferably the bacteriocin-producing microbial organism also produces a desired product. Preferably, the bacteriocin-producing microbial organism is a Gram-positive bacterium, for example a lactic acid bacterium, such as a Streptococcus species, such as Streptococcus salivarius. Preferably the undesired microbial organism is a pathogenic microbial organism or a contaminant.
In a seventh aspect, there is provided a method for identifying, selecting, and/or optimizing peptide ligands of peptide-responsive transcriptional regulators, comprising the following steps:
The present inventors have surprisingly developed a method, by which they have identified a group of peptide ligands inducing a unique peptide-responsive regulator of bacteriocin production. Particularly, as elaborated in the experimental part, the present inventors have surprisingly found that a peptide or peptidomimetic according to the invention can act as a ligand of the peptide-responsive regulators ScuR and/or SarF, thereby inducing the production of a group of class II type bacteriocins without necessarily inducing competence. Accordingly, the aspects and embodiments of the present invention as described herein solve at least some of the problems and needs as discussed herein.
In a first aspect, there is provided a peptide or peptidomimetic with a length of at least 6 residues comprising, consisting essentially of, or consisting of the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO:1), wherein:
The sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 may also be denoted as a sequence pattern or, simply, a sequence. A “sequence motif” or “sequence pattern” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to an amino-acid (or nucleotide) sequence that recurs, with a certain degree of variation, on several sites of a molecule or several different molecules and has, or is conjectured to have or is assumed to be linked to, a biological significance or exhibit a biological activity as described herein. A biological significance or biological activity of the peptide of the invention is preferably to be able to induce bacteriocin production in a microbial organism, more preferably without concomitantly inducing competence.
In some embodiments, a peptide or peptidomimetic according to the invention may have a length of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 residues, or at least 25 residues, or at least 30 residues, or at least 35 residues, or at least 40 residues, or at least 45 residues, or at least 50 residues. Accordingly, in other embodiments, a peptide or peptidomimetic according to the invention may have a minimal length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues and a maximal length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 residues.
As used herein, a “peptide” should be understood to encompass any peptide, regardless of whether it is generated through recombinant protein synthesis, purified from a native producer microbial organism or generated by means of chemical peptide synthesis. In some embodiments, the peptide comprises, consists essentially of, or consists of a non-naturally occurring amino acid sequence, a naturally occurring amino acid sequence, or a combination of these. Accordingly, there is also provided a vector comprising a nucleic acid encoding a peptide according to the invention. The term “vector” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a nucleic acid molecule, such as a plasmid, bacteriophage or animal virus, capable of introducing a heterologous nucleic acid sequence into a host cell. Further provided is a recombinant host cell comprising a vector according to the invention. The recombinant host cell can be a microbial host cell. Exemplary microbial host cells that can be used in this context are described later herein.
A “synthetic peptide” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a peptide which is generated by means of chemical peptide synthesis. Accordingly, in some embodiments, a peptide may be a synthetic peptide. A synthetic peptide according to the invention may be prepared or synthesized using conventional methods that are well known in the art. For instance, peptides can be synthesized by commonly used solid-phase synthesis methods such as those that involve a tert-butyloxycarbonyl protecting group (t-BOC) or fluorenylmethyloxycarbonyl protecting group (FMOC) for protection of alpha-amino groups. In such methods, amino acids are added sequentially to a growing amino acid chain. Such methods are, for instance, described in Merrifield (1963), J. Am. Chem. Soc. 85(14):2149-2154; and Atherton & Sheppard, Solid Phase Peptide Synthesis: A practical Approach (IRL Press, Oxford, UK, 1999), both of which are incorporated herein by reference. A peptide may further be modified by natural processes, such as post-translational processing, or by chemical modification techniques. Such modifications may be inserted in the peptide at any location, including in the backbone, amino acid side-chains and at the N- or C-terminus. Multiple types of modifications may occur in a single peptide, or a peptide may comprise several modifications of a single type. Illustrative but non-limiting examples of modifications are alkylation, acetylation, amidation, acylation, phosphorylation, methylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitination, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation, succinylation and sulfation.
As used herein, a “peptidomimetic” is understood to encompass all compounds whose essential elements (pharmacophore) mimic a natural peptide and which retain the ability to interact with the biological target and produce the same biological effect. In some embodiments, the peptidomimetic comprises, consists essentially of, or consists of a non-naturally occurring amino acid sequence. In an embodiment, the peptidomimetic does not occur in nature and is considered to be man-made. They typically arise either from modification of an existing peptide, or by designing similar systems that mimic peptides, such as peptoids and β-peptides. Structures and synthesis of peptidomimetics are for instance described in William D. Lubell (ed.), Peptidomimetics I and II, Topics in Heterocyclic Chemistry (Book 48), Springer 1st ed. 2017, XVI, 310 p, which is incorporated herein by reference. Modification of an existing peptide may be the result of natural processes, such as post-translational processing, or chemical modification techniques. In general, a peptidomimetic typically refers to a compound containing non-peptidic structural elements. Typical but non-limiting examples of non-peptidic structural elements are modifications of one or more existing amino acids, conformational restraints, cyclization of the polypeptide, isosteric replacement or other modifications. In some embodiments, a peptidomimetic may contain one or more or all substitutions of an amino acid by the corresponding D-amino acid. As used herein, “corresponding D-amino acid” denotes the D-amino acid counterpart of an L-amino acid. In some embodiments, a peptidomimetic may also contain non-natural amino acids. As used herein, “non-natural amino acid” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to non-genetically encoded amino acids, irrespective of whether they appear in nature or not. Non-natural amino acids that can be present in a peptidomimetic as described herein include: β-amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl-L-tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine; 4-tert-butyl hydrogen 2-azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; 2,4-diamino butyric acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2- aminopimelic acid; p-aminophenylalanine; 2,3-diaminobutyric acid; 2,3-diamino propionic acid; 2,2′-diaminopimelic acid; p-amino-L-phenylalanine; p-azido-L- phenylalanine; D-allyl glycine; p-benzoyl-L-phenylalanine; 3-benzothienyl alanine p-bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosine; epsilon-amino hexanoic acid; N-ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxy lysine; alio-hydroxy lysine; 3-(3-methyl-4-nitrobenzyl)-L-histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3- methyl-phenylalanine; N-methylglycine; N-methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; methionin sulfoxide; 2-napthylalanine; L-3-(2-naphthyl)alanine; isoserine; 3-phenylserine; norvaline; norleucine; 5,5,5-trifluoro-DL-leucine; ornithine; 3-chloro-tyrosine; N5-carbamoylornithine; penicillamine; phenylglycine; piperidinic acid; pyridylalanine; 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid; beta-2-thienylalanine; γ-carboxy-DL-glutamic acid; 4-fluoro-DL-glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy-tryptophan; 5-fluoro-tryptophan; 3-fluoro-valine. In some embodiments, a natural amino acid of a peptide or peptidomimetic according to the invention is substituted by a corresponding non-natural amino acid. As used herein, a “corresponding non-natural amino acid” refers to a non-natural amino acid that is a derivative of the reference natural amino acid. For instance, a natural amino acid can be substituted by the corresponding β-amino acid, which have their amino group bonded to the β-carbon rather than the α-carbon. According to some embodiments, a peptide or peptidomimetic of the invention can further be provided with a targeting moiety. It is known that peptidomimetics are able to circumvent some of the disadvantages associated with natural peptides: e.g. stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved.
As used herein, a “peptide or peptidomimetic” is understood to refer to a chain of a limited number of amino acids, generally between 2 and 50.
In a preferred embodiment, the sequence motif as described herein is located at the C-terminus of the peptide or peptidomimetic. In some other embodiments, the sequence motif as described herein may be located so that it is followed by 1, 2, 3, 4 or 5 additional C-terminal residues.
In some embodiments, a peptide or peptidomimetic as described herein does not comprise, consist essentially of, or consist of the amino acid sequence TNVTKSWWVLAGCNQWASNCNCGNVKGLT (SEQ ID NO: 751) as disclosed in U.S. Pat. No. 7,662,592.
In some embodiments, a peptide or peptidomimetic as described herein does not comprise, consist essentially of, or consist of the sequence VKGLT. In some embodiments, a peptide or peptidomimetic as described herein does not comprise a VKGLT sequence (SEQ ID NO: 752) at its C-terminus or at its C-terminus followed by 1, 2, 3, 4 or 5 additional C-terminal residues.
In some embodiments, a peptide or peptidomimetic according to the invention may be comprised in a polypeptide. In certain embodiments, the polypeptide can be processed by natural, chemical or biological peptide hydrolysis, including proteolysis mediated by recombinant or natural (endo- and exo-) proteases, in order to generate a peptide as described herein. Accordingly, in some embodiments there is provided a polypeptide able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising a peptide sequence as described herein, wherein the peptide or peptidomimetic can be released from the polypeptide by natural, chemical or biological peptide hydrolysis. Such polypeptide can be referred to as a “pro-polypeptide”.
In some embodiments a “pro-polypeptide”, i.e. a polypeptide able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising a peptide sequence as described herein may be a precursor peptide or peptidomimetic. Accordingly, in some embodiments there is provided a precursor peptide or peptidomimetic able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising a peptide or peptidomimetic as described herein, wherein the peptide or peptidomimetic can be released from the protein by natural, chemical or biological peptide hydrolysis. In some embodiments, a precursor peptide or peptidomimetic may comprise, consist essentially of, or consist of any of the sequences of SEQ ID NO: 3-24 and 749 followed by a tail of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional amino acids, for example a triple leucine tail. In some embodiments, a precursor peptide or peptidomimetic may comprise, consist essentially of, or consist of the sequence LAFWDSLGLLL (SEQ ID NO: 750).
In some embodiments, a polypeptide (“pro-polypeptide’) or a precursor peptide or peptidomimetic as described herein may have a length of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 residues. Accordingly, in other embodiments, a polypeptide (“pro-polypeptide”) or a precursor peptide or peptidomimetic as described herein may have a minimal length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 residues and a maximal length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 residues. In some embodiments, a polypeptide (“pro-polypeptide’) may have a length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 residues.
In some embodiments, processing occurs in or by a microbial cell. In other embodiments, processing occurs in an animal or human host. In certain embodiments, a peptide or peptidomimetic according to the invention may be comprised in a polypeptide which is a “pro-polypeptide”, further containing one or more other peptides with a specific activity separated by cleavage sites. As used herein, “cleavage site” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a polypeptide sequence that mediates the cleavage of a polypeptide (for example by hydrolysis of a peptide bond) to separate a single polypeptide into two or more discrete polypeptides. In some embodiments, a cleavage site comprises, consists essentially of, or consists of a consensus polypeptide sequence for cleavage by a “cleavage enzyme,” such as a peptidase. In some embodiments, the cleavage enzyme is a wild-type, a variant, or a synthetic cleavage enzyme, for example a wild-type, variant, or synthetic endopeptidase. In some embodiments, the cleavage sites are for a cleavage enzyme selected from the group consisting of: Arg-C proteinase, Asp-N endopeptidase, BNPS-Skatole, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Chymotrypsin-high specificity, Clostripain (Clostridiopeptidase B), CNBr, Enterokinase, Factor Xa, Formic acid, Glutamyl endopeptidase, GranzymeB, Hydroxylamine, lodosobenzoic acid, LysC, Neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoic acid), Pepsin (pH 1.3), Pepsin (pH>2), Proline-endopeptidase, Proteinase K, Staphylococcal peptidase I, Thermolysin, Thrombin, or Trypsin. The one or more other peptides with a specific activity can be signal molecules and/or quenching molecules and/or antimicrobial peptides and/or bacteriocins as described herein. Upon processing, such pro-polypeptide releases the peptide or peptidomimetic according to the invention as well as the one or more other peptides with a specific activity. A possible advantage of a peptide or peptidomimetic according to the invention when it is comprised in a precursor peptide or peptidomimetic or in a polypeptide and can be released by natural, chemical or biological peptide hydrolysis, is that the released peptides are able to to induce bacteriocins while precursor peptide or peptidomimetic or the full protein is not. Therefore, this constitutes an inducible or switchable system. In some embodiments, the inducible/switchable system may comprise, consist essentially of, or consist of a protease released in the environment (culture medium, microbiota, . . . ). In some embodiments, the inducible/switchable system may comprise, consist essentially of, or consist of a sensor protein directly or indirectly coupled to the activation of a transcriptional regulator. Activation of the transcriptional regulator would result in the net production/secretion of a protease that will cleave the precursor and release the active part of the inducibles peptide or peptidomimetic. The sensor may be capable to perceive stimuli from physico-chemical (temperature, pH, . . . ) changes in the culture condition or from the addition of biological or chemical compounds (preferentially sugar, peptides or peptidomimetics). Inducible promoters as described herein, such as xylose-, arabinose-, nickel-, nisin-, IPTG-, and pheromone-inducible promoters, may also be used. In some specific cases, the active peptide or peptidomimetic released might be the stimulus by itself, creating a positive feedback loop that promotes a rapid and sharp accumulation of the peptide or peptidomimetic in the environment (culture medium, microbiota, . . . ).
In a preferred embodiment, a peptide or peptidomimetic as described herein fulfills at least one or at least two or all of the following conditions:
In some embodiments, if all of the above-defined conditions are fulfilled, a peptide or peptidomimetic according to the invention may thus comprise, consist essentially of, or consist of the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Gly (SEQ ID NO: 25), wherein:
In some embodiments, Xaa2, Xaa3 and Xaa4 may be hydrophobic residues.
Sequence motifs may be represented by a consensus sequence: a single sequence consisting of the most commonly encountered residues at each site. Hence, a peptide or peptidomimetic according to an embodiment of the invention may comprise, consist essentially of, or consist of a sequence motif having the consensus sequence Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2). Thus, in some embodiments, a peptide or peptidomimetic according to the invention may comprise, consist essentially of, or consist of the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2). In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) may contain amino acid substitutions at 1, 2, 3, 4 or up to 5 positions. Amino acid substitutions can be conservative amino acid substitutions, as described herein. Examples of suitable amino acid substitutions in this context include the substitution of Phe for Tyr, Trp or His, and/or the substitution of Gly for Val or Ile. In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) may contain amino acid substitutions at 1, 2, 3, 4 or up to 5 positions, wherein Trp is not substituted. In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) contains amino acid substitutions at 1, 2, 3, 4 or up to 5 positions, wherein Trp is not substituted and Gly is substituted by Val or Ile. In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) may contain amino acid substitutions at 1, 2, 3 or up to 4 positions, wherein Trp and Gly are not substituted. In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) may contain amino acid substitutions at 1, 2, 3 or up to 4 positions, wherein Trp and Gly are not substituted and Phe is substituted by Tyr, Trp or His. In some embodiments, the sequence motif Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2) may contain amino acid substitutions at 1, 2, or up to 3 positions, wherein Trp, Gly and Phe are not substituted.
It will be appreciated that a consensus sequence can also be represented by a sequence logo. A sequence logo is a graphical representation of the consensus sequence, in which the size of a symbol is related to the frequency that a given nucleotide or amino acid occurs at a certain position. The more conserved the residue, the larger the symbol for that residue is drawn; the less conserved the residue, the smaller the symbol is drawn. Sequence logos can for example be generated using WebLogo, available at on the world wide web at weblogo.berkely.edu. In some embodiments, the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 as described herein, can be represented by a sequence logo as depicted in
According to a preferred embodiment, a peptide or peptidomimetic as described herein comprises, consists essentially of, or consists of one of the following sequences:
In some embodiments, there is also provided a peptide or peptidomimetic which comprises, consists essentially of, or consists of a sequence in which 1, 2, 3, 4 or up to 5 residues are deleted, added or substituted compared to any of the sequences of SEQ ID NO's: 3-24 and 749. Amino acid substitutions can be conservative amino acid substitutions, as described herein.
In a preferred embodiment, a peptide or peptidomimetic according to the invention is able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence.
Natural DNA transformation, or just “transformation”, refers to a lateral gene transfer mechanism during which bacteria take up naked DNA from their environment and stably integrate it in their genome. The physiological state during which bacteria become able to take up DNA is named competence. Although natural transformation drives genome plasticity and adaptability, competence induction may also be associated with several disadvantages. For example, competence induction is likely to cause deleterious effects in the chromosome of the recipient bacteria and negatively impact cell growth by imposing an important energy burden on the recipient cells (Fontaine et al. Infection, Genetics and Evolution 2015, 33:343-360). Competence is also associated with a suppression of basal metabolism, which may have consequences for the microbe's resilience to fluctuations in the environment, as competence is costly in terms of use of energy and protein translation (Zaccaria et al. Plos One 2016, 11(5):e0153571). Also, competence induction may contribute to the unwanted spread of antibiotic resistance or bacteriocin immunity genes. The competence window is thus generally tightly regulated in response to species-specific environmental conditions or signaling oligopeptides called competence pheromones and limited to a proportion of the cell population.
In some embodiments, a peptide or peptidomimetic according to the invention is able to induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, by inducing a peptide-responsive regulator that regulates genes involved in the synthesis of bacteriocins. Within the context of this disclosure, “inducing bacteriocin production” may be assessed by a skilled person at the nucleic acid level and/or at the amino acid level using common known techniques. As soon as a bacteriocin is detectable using common known techniques, the peptide or peptidomimetic according to embodiments of the invention will be said to exhibit a biological activity. A bacteriocin may be detectable using common known techniques after 1, 2, 3, 5, 10, 20, 30, 45, 60 or 90 minutes or also after 2, 3, 4, 5, 6, 8, 10, 12, 16, 20 or 24 hours, including intervals between any two of the listed values. In a preferred embodiment, the peptide-responsive regulator that regulates genes involved in the synthesis of bacteriocins does not regulate genes involved with competence, such as the central regulator of competence ComX (also denoted alternative sigma factor X, SigX or σX), ComK, SigH (also denoted alternative sigma factor H or σH) and TfoX (also denoted Sxy). As used herein, regulation includes both direct regulation as well as indirect regulation. Preferred peptide-responsive regulators regulate genes involved in the synthesis of bacteriocins but do not regulate genes involved with competence, such as ScuR and SarF.
A peptide or peptidomimetic as described herein is a ligand of a peptide-responsive regulator, preferably a peptide-responsive regulator that regulates genes involved in the synthesis of bacteriocins. Examples of suitable peptide-responsive regulators in this context are RRNPP type peptide-responsive regulators, such as ScuR and SarF.
Exemplary bacteriocins in this context are bacteriocins as described herein. Preferred bacteriocins in this context comprise, consist essentially of, or consist of class II bacteriocins, for example salivaricins.
In a further aspect, there is provided a culture medium comprising a peptide or peptidomimetic as described herein, and/or a polypeptide as described herein. A culture medium can be either a liquid culture medium or a solid culture medium, such as an agar-based solid culture medium.
A preferred culture medium as described herein induces bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, when the microbial organism is cultured in the culture medium. Exemplary bacteriocins in this context are bacteriocins as described herein. Preferred bacteriocins in this context comprise, consist essentially of, or consist of class II bacteriocins, for example salivaricins.
Typically, a culture medium as described herein comprises a peptide or peptidomimetic as described herein in a concentration ranging from 1 nM (0.001 μM) to 10 μM. Thus, a suitable concentration may be at least 0.001 μM, at least 0.01 μM, at least 0.1 μM, at least 1 μM or at least 10 μM. Accordingly, in other embodiments, a suitable concentration may lie between a minimum of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM and a maximum of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM.
In some embodiments, a suitable concentration is a concentration which induces bacteriocin production in a microbial organism that is cultured in the culture medium. A skilled person knows how to determine such suitable concentrations, for example based on an assay as used in the experimental part.
In some embodiments, the culture medium further comprises a signal molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin as described herein.
In a further aspect, there is provided a composition comprising a peptide or peptidomimetic as described herein, and/or a polypeptide as described herein, and a solvent.
Suitable solvents include any solvent or mixture of solvents in which a peptide or peptidomimetic or polypeptide as described herein can be dissolved at a suitable concentration.
The number and types of ionic charges in the peptide determine its solubility in aqueous solutions. In general, the more charged residues the peptide possesses, the more soluble it is in aqueous solutions. In addition, peptides generally have more charges at pH 6-8 than at pH 2-6. It is for this reason that peptides are better dissolved at near neutral pH. Among the many exceptions to the rule are peptide sequences that are very hydrophobic and those that tend to aggregate. While the hydrophobicity of the sequence is the primary cause of aggregation, peptides can also aggregate or “gel” through extensive hydrogen bonding network. Non-limiting examples of solvents that can be used in the context of the invention are water, ethanol, ammoniumhydroxide, dimethylsulfoxide (DMSO), acetic acid, acetonitrile and dimethylformamide (DMF). Dissolution can be enhanced by sonication.
A peptide or peptidomimetic or polypeptide according to the invention exhibits a number of activities that can be advantageously used in a wide range of applications, including therapeutic or agricultural applications, and applications in probiotics, cosmetology, cleaning of surfaces (including surfaces of chemically fragile medical devices), biotechnology, biofermentation processes, and food preservation. Provided therefore are compositions comprising a peptide or peptidomimetic or polypeptide as described herein or an acceptable salt thereof, and an acceptable carrier, diluent and/or excipient. Each of the acceptable salt, carrier, diluent and/or excipient can be a salt, carrier, diluent and/or excipient which is suitable for the intended use or application, for example pharmaceutical or agricultural.
A peptide or peptidomimetic or polypeptide according to the invention exhibits a number of activities that can be advantageously used in agricultural applications. Provided therefore are agricultural compositions comprising a peptide or peptidomimetic or polypeptide as described herein or an agriculturally acceptable salt thereof, and an agriculturally acceptable carrier, diluent and/or excipient.
Pharmaceutically and/or agriculturally acceptable salts include, but are not limited to, acid addition salts and base addition salts. As used herein, “pharmaceutically acceptable salt” of a peptide refers to a salt that retains the desired function of the peptide, and is suitable for administration to humans or animals. As used herein, “agriculturally acceptable salt” of a peptide refers to a salt that retains the desired function or activity of the peptide at least to some extent, and is suitable for use in the environment including animals, plants, water, air and/or soil. Within the context of the application, “at least to some extent” means that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the initial desired function or activity is retained. Methods for the preparation of salts of peptides are known in the art and generally involve mixing of the peptide with a pharmaceutically and/or agriculturally acceptable acid or base, for instance, by reacting the free acid or free base forms of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed by vacuum or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin. Non-limiting examples of pharmaceutically and/or agriculturally acceptable acids and bases are organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid, and thiocyanic acid, which form ammonium salts with free amino groups of polypeptides, and bases that form carboxylate salts with free carboxylic groups of polypeptides, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di- and trialkylamines, and arylamines.
Preferred compositions, pharmaceutical compositions and agricultural compositions as described herein induce bacteriocin production in a microbial organism, preferably without concomitantly inducing competence. Thus, compositions, pharmaceutical compositions and agricultural compositions as described herein include compositions, pharmaceutical compositions and agricultural compositions for inducing bacteriocin production in a microbial organism, preferably without concomitantly inducing competence. Preferred bacteriocins in this context comprise, consist essentially of, or consist of class II bacteriocins, for example salivaricins.
Typically, compositions, pharmaceutical compositions and agricultural compositions as described herein comprise a peptide or peptidomimetic or polypeptide as earlier described herein at a concentration ranging from 0.001 to 10 μM. Thus, a suitable concentration may be at least 0.001 μM, at least 0.01 μM, at least 0.1 μM, at least 1 μM or at least 10 μM. Accordingly, in other embodiments, a suitable concentration may lie between a minimum of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM and a maximum of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM.
In some embodiments, a suitable concentration is a concentration which induces bacteriocin production in a microbial organism when the composition is administered to the microbial organism. A skilled person knows how to determine such suitable concentrations, for example based on an assay as used in the experimental part.
In some embodiments, the composition further comprises a signal molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin.
Within the context of culture media and compositions and methods according to embodiments of the invention, a “signal molecule” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a secreted or released molecule that is capable of modulating, inducing, or inhibiting an activity or process in the cell that produced it, or in a different cell (a subject cell can be a microbial cell or a non-microbial cell, for example a cell of a multicellular organism such as an animal or plant).
In some embodiments, a signal molecule may be selected from the group consisting of: quorum sensing molecules, signal transduction receptor ligands, growth factors, hormones, and cytokines. A signal molecule as described herein can be wild-type, mutant, or synthetic. Examples of suitable signal molecules include quorum sensing peptides (also called pheromones). In some embodiments, a signal molecule comprises, consists essentially of, or consists of a signaling peptides, quorum sensing molecules (for example, quorum sensing peptides), signal transduction receptor ligands, growth factors, hormones, or cytokine. In some embodiments, a signal molecule comprises, consists essentially of, or consists of a combination of two or more of signaling peptides, quorum sensing molecules (for example, quorum sensing peptides), signal transduction receptor ligands, growth factors, hormones, and cytokines, which can include combinations of two or more of the same type of molecule (for example a combination of two signaling peptides or a combination of two receptor ligands), as well as combinations of two or more different kinds of molecules (e.g., a combination of a cytokine and a hormone). In some embodiments, the signal molecule stimulates, inhibits, increases, or decreases the production of bacteriocins and/or the growth rate of a subpopulation of a microbiota. In some embodiments, the signal molecule comprises, consists essentially of, or consists of a quorum sensing peptide, or a variant thereof as described herein.
Examples of quorum sensing peptides suitable for culture media and compositions according to embodiments of the invention include, but are not limited to, quorum sensing peptides such as the peptides shown in Table 1 below, including variants of these peptides, and combinations of two or more of any of these peptides.
In some embodiments, the quorum sensing peptides are naturally-occuring. In some embodiments, the quorum sensing peptide comprises, consists essentially of, or consists of a variant of a naturally-occurring quorum sensing peptide. In some embodiments, the quorum sensing peptide comprises, consists essentially of, or consists of a synthetic peptide. Information on quorum sensing peptides, including example sequences, can be found on the quorumpeps database, accessible on the world wide web at quorumpeps.ugent.be, which is hereby incorporated by reference in its entirety.
Bacillus anthracis
Bacillus anthracis
Bacillus anthracis
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus cereus, Bacillus thuringiensis
Bacillus halodurans
Bacillus halodurans
Bacillus pumilus
Bacillus stearothermophilus
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus thuringiensis
Carnobacterium maltaromaticum
Carnobacterium maltaromaticum
Carnobacterium piscicola
Carnobacterium piscicola,
Carnobacterium maltaromaticum
Clostridium acetobutylicum
Clostridium acetobutylicum
Clostridium acetobutylicum
Clostridium botulinum
Clostridium botulinum
Clostridium botulinum
Clostridium botulinum
Clostridium botulinum
Clostridium perfringens
Clostridium perfringens
Clostridium perfringens
Clostridium perfringens
Clostridium sporogenes
Clostridium sporogenes
Clostridium thermocellum
Cryptococcus neoformans
Cryptococcus neoformans
Cryptococcus neoformans
Eikenella corodens
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
Escherichia coli
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus sakei
Lactobacillus sakei
Lactobacillus sakei
Lactococcus lactis
Listeria monocytogenes
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Staphylococcus arlettae
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus auricularis
Staphylococcus auricularis
Staphylococcus capitis
Staphylococcus capitis
Staphylococcus caprae
Staphylococcus caprae
Staphylococcus caprae
Staphylococcus carnosus
Staphylococcus cochnii subsp. cochnii
Staphylococcus cochnii subsp.
urealyticum
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus gallinarum
Staphylococcus lugdunensis
Staphylococcus lugdunensis
Staphylococcus simulans
Staphylococcus simulans
Staphylococcus simulans
Staphylococcus warneri
Staphylococcus xylosus
Streptococcus agalactiae
Streptococcus dysgalactiae
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptomyces species
Thermotoga maritima
Within the context of culture media and compositions and methods according to embodiments of the invention, a “quenching molecule” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to (peptidic) quenching molecules that prevent the signal molecules to reach their cognate receptors by enzymatic activity such as proteolysis, addition of inactivating chemical groups or by competition with the signal molecule.
Within the context of culture media and compositions and methods according to embodiments of the invention, an “antimicrobial peptide” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a class of peptides that confer innate immune activity to kill or arrest the growth of microbial organisms. Classically, antimicrobial peptides have been described as peptides produced by the innate immune systems of invertebrates and vertebrates. Thus, while bacteriocins have classically been referred to a class of microbial gene products that target microbial organisms, antimicrobial peptides have classically been referred to as a class of invertebrate and vertebrate gene products that target microbial organisms.
Examples of antimicrobial peptides suitable for peptides or peptidomimetics, culture media, compositions, methods and uses according to the invention are known in the art, and can be found, for example, at The Antimicrobial Peptide Database accessible on the world wide web at aps.unmc.edu/AP/main.php, which is incorporated herein by reference in its entirety. Over 1000 antimicrobial peptides and variants thereof have been identified and cataloged. The Antimicrobial Peptide Database is described in Wang et al. (2016), Nucleic Acids Res. 44:D1087-D1093, which is incorporated herein by reference in its entirety.
Over 1000 antimicrobial peptides and variants thereof have been identified and cataloged. The Antimicrobial Peptide Database is described in Wang et al. (2016), Nucleic Acids Res. 44 (Database issue): D1087-D1093, which is incorporated herein by reference in its entirerty. Examples of antimicrobial peptides include bacteriocins, antibacterial, antiviral, anti-HIV, antifungal, antiparasitic and anticancer peptides, such as Dermaseptin-B2, Abaecin, Ct-AMPI, Andropin, Aurein 1.1, Lactofericin B, and Heliomicin. Methods, culture media, and compositions of some embodiments comprise naturally occuring antimicrobial peptides, or a nucleic acid encoding the same, and/or nonnaturally occurring antimicrobial peptides, or a nucleic acid encoding the same. Methods, culture media, and compositions of some embodiments include antimicrobial peptides that comprise a mutation or variation in a naturally-occuring antimicrobial peptide, or a nucleic acid encoding the same. Methods, culture media, and compositions of some embodiments comprise antimicrobial peptides comprising, consisting essentially of, or consisting of non-naturally occuring peptide sequences, or nucleic acids encoding the same.
It is further contemplated that methods, culture media, and compositions of some embodiments herein can be in conjunction with naturally occurring antimicrobial peptides, variants of naturally occurring antimicrobial peptides, and/or synthetic antimicrobial peptides. As such, antimicrobial peptides of methods, culture media, and compositions of some embodiments can comprise, consist essentially of, or consist of naturally occurring antimicrobial peptides, variants of naturally occurring antimicrobial peptides, and/or synthetic antimicrobial peptides. In some embodiments, a variant antimicrobial peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a reference antimicrobial peptide (for example Dermaseptin-B2, Abaecin, Ct-AMPI, Andropin, Aurein 1.1, Lactoferricin B, or Heliomicin).
In a further aspect, there is provided a microbial organism able to produce and/or secrete a peptide or peptidomimetic and/or a precursor peptide or peptidomimetic and/or a protein as described herein. A variety of bacterial species and strains can be used in accordance with embodiments herein, as explained in the section “General descriptions”. In some embodiments, the microbial organism is a Gram-positive bacterium, preferably a lactic acid bacterium. In some embodiments, the microbial organism is a Streptococcus sp., preferably Streptococcus salivarius. In some embodiments, the microbial organism is “generally recognized as safe” (GRAS). In some embodiments, the microbial organism is a commensal bacterium from the microbiota.
In a further aspect, there is provided a method for inducing bacteriocin production in a microbial organism, preferably without concomitantly inducing competence, comprising administering a peptide or peptidomimetic or polypeptide as described herein to the microbial organism, and/or culturing the microbial organism in a culture medium as described herein, and/or administering a composition as described herein to the microbial organism. In some embodiments of a method according to the invention, administering a peptide or peptidomimetic or polypeptide as described herein to the microbial organism and administering a composition as described herein to the microbial organism may optionally be followed by a culturing step. In some embodiments, a peptide or peptidomimetic or polypeptide or a composition as described herein can be administered to a microbial organism in a host, such as a patient having an infection or an imbalance in the microbiome.
Also provided is a use of a peptide or peptidomimetic or polypeptide as described herein, a culture medium as described herein, or a composition as described herein for inducing bacteriocin production in a microbial organism. The use can be without concomitantly inducing competence.
In a further aspect, there is provided a method for inducing bacteriocin production in a first microbial organism, preferably without concomitantly inducing competence, comprising administering to the first microbial organism a second microbial organism able to produce and/or secrete a peptide or peptidomimetic and/or a polypeptide as described herein and/or co-culturing the first microbial organism with the second microbial organism able to produce and/or secrete a peptide or peptidomimetic and/or a polypeptide as described herein.
Also provided is a use of a microbial organism able to produce and/or secrete a peptide or peptidomimetic and/or a polypeptide as described herein fo inducing bacteriocin production in a microbial organism. The use can be without concomitantly inducing competence.
In some embodiments of a method and/or a use as described herein, the bacteriocin production in the microbial organism neutralizes a second, undesired microbial organism.
As used herein, “neutralizes” and variations to this root term has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It includes any form of inhibition or arrest of microbial growth and/or division (bacteriostatic effect), as well as any cytotoxic or bactericidal effect (killing). Neutralization can be fully or partially. For example, the whole population or only a part of the targeted population may be growth-inhibited or killed. More particularly, partial neutralization may mean that at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the microbial organisms is growth-inhibited or killed. A skilled person knows how to assess growth inhibition and killing, for example based on assays that are used in the experimental part. “Undesired” microbial organism as used herein refers to any microbial organism that is targeted for neutralization. For example, an undesired microbial organism may be a pathogenic microbial organism or a contaminant.
In some embodiments of a method and/or a use as described herein, the microbial organism that produces bacteriocin is a bacterium. In some embodiments, the microbial organism that produces bacteriocin is a Gram-negative bacterium. According to some embodiments, the microbial organism that produces bacteriocin is a Gram-positive bacterium, for example a lactic acid bacterium, such as a Streptococcus species, such as Streptococcus salivarius.
In the context of a method and/or a use according to the invention, the undesired microbial organism may be a pathogenic microbial organism, preferably a pathogenic microbial organism that is susceptible to neutralization by a bacteriocin as described herein. In some embodiments, the undesired microbial organism is a Gram-positive bacterium. In still some embodiments, the undesired microbial organism is a pathogenic Gram-positive bacteria. The pathogenic Gram-positive bacteria can be selected from the group consisting of the following genera: Staphylococcus, Enterococcus, Streptococcus, Listeria, Bacillus, Brochothrix, Clostridium, Mycobacterium, Propionibacterium, or Corynebacterium. Accordingly, Gram-positive bacterial species may be Staphylococcus aureus, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Staphylococcus epidermidis, Clostridium difficile, Clostridium perfringens, Mycobacterium tuberculosis, Propionibacterium acnes, and Corynebacterium diphteriae.
In some embodiments, a method and/or a use as described herein may be a method and/or a use wherein the microbial organism belongs to the microbiota. In other words, the microbial organism that produces bacteriocin is a microbial organism that belongs to the microbiota. In this context, a microbiota can be the microbiota of human, for example the microbiota of the skin and/or the gut and/or the mouth and/or the vagina, the microbiota of an animal, for example the microbiota of the skin and/or the gut and/or the mouth and/or the vagina, and/or the microbiota of a plant, for example the microbiota of the roots.
In other embodiments, a method and/or a use as described herein may be a method and/or a use wherein the microbial organism produces a desired product. In other words, the microbial organism that produces bacteriocin is a microbial organism that also produces a desired product. In this context, the microbial organism can exist in a number in commercially useful environments such as industrial cultures, fermenters, pharmaceutical, biological, and cosmetic manufacturing and in products, such as foods (for human and/or animals), drug products, and cosmetic products. Hence, in some embodiments, the undesired microbial organism may be a contaminant in commercially useful environments such as industrial cultures, fermenters, pharmaceutical, biological, and cosmetic manufacturing and in products, such as foods (for human and/or animals), drug products, and cosmetic products.
Exemplary bacteriocins in this context are bacteriocins as described herein. In the context of a method and/or a use according to the invention, the bacteriocin may be a class II bacteriocin, for example salivaricin.
In a further aspect, there is provided a method for identifying, selecting and/or optimizing peptide ligands of peptide-responsive transcriptional regulators, comprising the following:
In a preferred embodiment, there is provided a method for identifying, selecting and/or optimizing peptide ligands of peptide-responsive transcriptional regulators, comprising the following steps:
In a preferred embodiment, the peptide-responsive transcriptional regulator belongs to the RRNPP family. RRNPP proteins are named after the different sensors described: Rgg, Rap, NprR, PlcR, and PrgX. They are characterized at the structural level by tetratricopeptide repeat (TPR) domains, which are involved in the regulator/peptide interaction.
In a preferred embodiment, the peptide responsive regulator is a regulator of bacteriocin synthesis. The peptide responsive regulator of bacteriocin synthesis may be ScuR or SarF.
In a preferred embodiments, a method as described herein may be a method wherein the nucleotide sequences of the randomized genes comprise both fixed-sequence codons and degenerate codons. As used herein, degenerate codons include mixtures of nucleotides at one, two or three positions and are used for introducing random diversity in the genes during oligonucleotide synthesis. For example, the complete set of standard amino acids can be encoded using NNK or NNS codons, where N=A or T or G or C, K=G or T and S=C or G.
In some embodiments, the randomized genes are optionally further operably linked to a selectable marker. Accordingly, the transforming step of a method as described herein optionally involves a selection step for successful transformants by selecting or enriching transformants in the presence of the selectable marker.
In some embodiments, the selectable marker encoded by the microbial organism is different from the selectable marker operably linked to the randomized genes. Exemplary selectable markers useful in some embodiments herein are antibiotic-resistance conferring genes such as cat (chloramphenicol acetyl transferase), erm (erythromycin ribosome methylation) and spec (spectinomycine resistance gene). Other suitable selectable markers may be bacteriocin immunity genes.
Inducible promoters are promoters which drive transcription only in the presence of a suitable inducer molecule. Inducible promoters useful in some embodiments herein are xylose-, arabinose-, nickel-, nisin-, IPTG-, and pheromone-inducible promoters. More exemplary promoters suitable for embodiments herein are given in Tables 2 and 3 below.
In some embodiments, the randomized genes may also be operably linked to a sequence which is homologous to a sequence of the microbial organism's genome to allow recombination at a desired locus in the microbial organism's genome. In some embodiments, the homologous sequence has a length of 50-2000 nucleotides, for example 100-1000 nucleotides, 200-800 nucleotides, or 500 nucleotides. In some embodiments of a method as described herein, the microbial organism that encodes a selectable marker is a bacterium. In some embodiments, the microbial organism that encodes a selectable marker is a Gram-negative bacterium. According to some embodiments, the microbial organism that encodes a selectable marker is a Gram-positive bacterium, for example a lactic acid bacterium, such as a Streptococcus species, such as Streptococcus salivarius.
Within the context of peptides or peptidomimetics, polypeptides, culture media, compositions, microbial organisms, and methods and uses according to embodiments of the invention, a “bacteriocin” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). A bacteriocin can neutralize at least one cell other than the individual host cell in which the polypeptide is made, including cells clonally related to the host cell and other microbial cells. Neutralization can be fully or partially as described herein. As used herein, bacteriocin also encompasses a cell-free or chemically synthesized version of such a polypeptide. A synthetic variant of a bacteriocin may be derived from the bacteriocin secreted by a host cell in any way as long as the synthetic variant still exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the activity of the corresponding bacteriocin secreted by a host cell.
Detailed descriptions of bacteriocins, including methods and compositions for using bacteriocins to control the growth of microbial cells can be found, for example, in U.S. Pat. No. 9,333,227, which is hereby incorporated by reference. “Bacteriocin” is not limited by the origin of the polypeptide, and by way of example is contemplated to encompass any bacteriocin, such as naturally-occurring bacteriocins, synthetic bacteriocins, and variants and combinations thereof.
A cell that expresses a particular “immunity modulator” is immune to the neutralizing effects of a particular bacteriocin or group of bacteriocins. As such, bacteriocins can neutralize a cell producing the bacteriocin and/or other microbial cells, so long as these cells are “susceptible”, i.e. do not produce an appropriate immunity modulator. As such, a bacteriocin can exert cytotoxic or growth-inhibiting effects on a plurality of other microbial organisms. In an embodiment, a bacteriocin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell. In another embodiment, a bacteriocin is chemically synthesized. Some bacteriocins can be derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example processing by a protease) to yield the polypeptide of the bacteriocin itself. As such, in some embodiments, a bacteriocin is produced from a precursor polypeptide. In some embodiments, a bacteriocin comprises, consists essentially of, or consists of a polypeptide that has undergone post-translational modifications, for example cleavage, or the addition of one or more functional groups.
Neutralizing activity of bacteriocins can include inhibition or arrest of microbial growth and/or division, or cytotoxicity. Some bacteriocins have cytotoxic activity (e.g. “bacteriocide” effects), and thus can kill microbial organisms, for example bacteria, yeast, algae, synthetic micoorganisms, and the like. Some bacteriocins can inhibit the growth and/or division of microbial organisms (e.g. “bacteriostatic” effects), for example bacteria, yeast, algae, synthetic microorganisms, and the like, for example by arresting the cell cycle.
A number of bacteriocins have been identified and characterized (see Table 4 and 5). Without being limited by any particular theory, exemplary bacteriocins can be classified as “class I” bacteriocins, which typically undergo post -translational modification, and “class II” bacteriocins, which are typically unmodified. Additionally, exemplary bacteriocins in each class can be categorized into various subgroups, as summarized in Cotter, P. D. et al. “Bacteriocins—a viable alternative to antibiotics” Nature Reviews Microbiology 11(2): 95-105, hereby incorporated by reference. Without being limited by any particular theory, bacteriocins can effect neutralization of a target microbial cell in a variety of ways. For example, a bacteriocin can permeabilize a cell wall, thus depolarizing the cell wall and interfering with respiration.
A number of bacteriocins can be used in accordance with embodiments herein. Exemplary bacteriocins are shown in Table 5. In some embodiments, at least one bacteriocin comprising, consisting essentially of, or consisting of a polypeptide sequence of Table 5 is provided. As shown in Table 5, some bacteriocins function as pairs of molecules. As such, it will be understood that unless explicity stated otherwise, when a functional “bacteriocin” or “providing a bacteriocin,” or the like is discussed herein, functional bacteriocin pairs are included along with bacteriocins that function individually. With reference to Table 5, “organisms of origin” listed in parentheses indicate alternative names and/or strain information for organisms known to produce the indicated bacteriocin.
Embodiments herein also include peptides and proteins with identity to bacteriocins described in Table 5. The term “identity” is meant to include nucleic acid or protein sequence homology or three-dimensional homology. Several techniques exist to determine nucleic acid or polypeptide sequence homology and/or three-dimensional homology to polypeptides. These methods are routinely employed to discover the extent of identity that one sequence, domain, or model has to a target sequence, domain, or model. A vast range of functional bacteriocins can incorporate features of bacteriocins disclosed herein, thus providing for a vast degree of identity to the bacteriocins in Table 5. In some embodiments, a bacteriocin has at least 50% identity, for example, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the polypeptides of Table 5.
While the bacteriocins in Table 5 are naturally-occurring, the skilled artisan will appreciate that variants of the bacteriocins of Table 5, naturally-occurring bacteriocins other than the bacteriocins of Table 5 or variants thereof, or synthetic bacteriocins can be used according to some embodiments herein. In some embodiments, such variants have enhanced or decreased levels of cytotoxic or growth inhibition activity on the same or a different microorganism or species of microorganism relative to the wild type protein. Several motifs have been recognized as characteristic of bacteriocins. For example, the motif YGXGV (SEQ ID NO: 236), wherein X is any amino acid residue, is an N-terminal consensus sequence characteristic of class IIa bacteriocins. Accordingly, in some embodiments, a synthetic bacteriocin comprises an N-terminal sequence with at least 50% identity to SEQ ID NO: 236, for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 236. In some embodiments, a synthetic bacteriocin comprises a N-terminal sequence comprising SEQ ID NO: 236. Additionally, some class IIb bacteriocins comprise a GXXXG motif (SEQ ID NO: 237) (X means any amino acid). Without being limited by any particular theory, it is believed that the GXXXG (SEQ ID NO: 237) motif can mediate association between helical proteins in the cell membrane, for example to facilitate bacteriocin-mediated neutralization through cell membrane interactions. As such, in some embodiments, the bacteriocin comprises a motif that facilitates interactions with the cell membrane. In some embodiments, the bacteriocin comprises a GXXXG (SEQ ID NO: 237) motif. Optionally, the bacteriocin comprising a GXXXG (SEQ ID NO: 237) motif can comprise a helical structure. In addition to structures described herein, “bacteriocin” as used herein also encompasses structures that have substantially the same effect on microbial cells as any of the bacteriocins explicitly provided herein.
It has been shown that fusion polypeptides comprising, consisting essentially of, or consisting of two or more bacteriocins or portions thereof can have neutralizing activity against a broader range of microbial organisms than either individual bacteriocin. For example, it has been shown that a hybrid bacteriocin, Ent35-MccV (GKYYGNGVSCNKKGCSVDWGRAIGIIGNNSAANLATGGAAGWKSGGGASGRD IAMAIGTLSGQFVAGGIGAAAGGVAGGAIYDYASTHKPNPAMSPSGLGGTIKQKP EGIPSE AWNYAAGRLCNWSPNNLSDVCL, SEQ ID NO: 238) displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria (Acuna et al. (2012), FEBS
Open Bio, 2: 12-19). It is noted that that Ent35-MccV fusion bacteriocin comprises, from N -terminus to C-terminus, an N-terminal glycine, Enterocin CRL35, a linker comprising three glycines, and a C-terminal Microcin V. It is contemplated herein that bacteriocins can comprise fusions of two or more polypeptides having bacteriocin activity. In some embodiments, a fusion polypeptide of two or more bacteriocins is provided. In some embodiments, the two or more bacteriocins comprise, consist essentially of, or consist of polypeptides from Table 5, or modifications thereof. In some embodiments, the fusion polypeptide comprising of two or more bacteriocins has a broader spectrum of activity than either individual bacteriocin, for example having neutralizing activity against more microbial organisms, neutralizing activity under a broader range of environmental conditions, and/or a higher efficiency of neutralization activity. In some embodiments, a fusion of two or more bacteriocins is provided, for example two, three, four, five, six, seven, eight, nine, or ten bacteriocins. In some embodiments, two or more bacteriocin polypeptides are fused to each other via a covalent bond, for example a peptide linkage. In some embodiments, a linker is positioned between the two bacteriocin polypeptides. In some embodiments, the linker comprises, consists essentially of, or consists of one or more glycines, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glycines. In some embodiments, the linker is cleaved within the cell to produce the individual bacteriocins included in the fusion protein. In some embodiments, a bacteriocin as provided herein is modified to provide a desired spectrum of activity relative to the unmodified bacteriocin. For example, the modified bacteriocin may have enhanced or decreased activity against the same organisms as the unmodified bacteriocin. Alternatively, the modified bacteriocin may have enhanced activity against an organism against which the unmodified bacteriocin has less activity or no activity.
Lactobacillus acidophilus
Lactobacillus acidophilus
Lactobacillus acidophilus
Lactobacillus gasseri
Staphylococcus aureus
Enterococcus avium (Streptococcus
avium)
Enterococcus faecalis
Lactococcus lactis
Enterococcus faecium
Clostridium botulinum
Streptococcus equinus
Brochothrix campestris
Butyrivibrio fibrisolvens
Butyrivibrio fibrisolvens
Carnobacterium maltaromaticum
Carnobacterium maltaromaticum
Carnobacterium maltaromaticum
Carnobacterium maltaromaticum
Pectobacterium carotovorum subsp.
carotovorum (Erwinia carotovora
Bacillus cereus
Streptoverticillium
griseoverticillatum
Geobacillus kaustophilus (strain
Clostridium tyrobutyricum
Bacillus coagulans
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Enterococcus columbae
Lactobacillus curvatus
Streptomyces sp.
Bacillus halodurans (strain ATCC
Carnobacterium divergens
Carnobacterium divergens
Carnobacterium divergens
Enterococcus durans
Enterococcus durans
Streptococcus dysgalactiae subsp.
equisimilis (Streptococcus
equisimilis)
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis (strain ATCC
Enterococcus faecium
Enterococcus faecalis
Enterococcus faecium
Enterococcus mundtii
Enterococcus faecalis
Enterococcus faecium
Enterococcus faecium
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
Enterococcus faecium
Enterococcus faecalis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus gallinarum
Lactococcus garvieae
Lactococcus garvieae
Lactobacillus gasseri
Lactobacillus gasseri
Lactobacillus plantarum
Haloferax mediterranei (strain
Haloarchaeon S8a
Lactobacillus helveticus
Enterococcus hirae
Lactobacillus johnsonii (strain
Lactobacillus johnsonii (strain
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. lactis
Lactococcus lactis
Lactococcus lactis
Lactobacillus amylovorus
Lactobacillus sakei L45
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. cremoris
Lactococcus lactis subsp. cremoris
Lactococcus sp. QU 12
Brevibacillus sp. GI-9
Leuconostoc pseudomesenteroides
Leuconostoc pseudomesenteroides
Leuconostoc gelidum
Leuconostoc carnosum
Leuconostoc mesenteroides
Bacillus licheniformis (strain DSM
Brevibacterium linens
Listeria innocua
Bacillus sp. (strain HIL-Y85/54728)
Leuconostoc mesenteroides
Clavibacter michiganensis subsp.
michiganensis
Escherichia coli
Escherichia coli
Klebsiella pneumoniae
Escherichia coli
Escherichia coli
Escherichia coli
Enterococcus mundtii
Enterococcus mundtii
Streptococcus mutans
Streptococcus mutans
Lactococcus lactis subsp. lactis
Lactococcus lactis
Lactococcus lactis
Streptococcus uberis
Lactococcus lactis subsp. lactis
Staphylococcus warneri
Paenibacillus polymyxa (Bacillus
polymyxa)
Pediococcus acidilactici
Pediococcus pentosaceus (strain
Staphylococcus epidermidis
Carnobacterium maltaromaticum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum (strain
Propionibacterium jensenii
Propionibacterium thoenii
Propionibacterium freudenreichii
Pseudomonas aeruginosa
Pseudomonas aeruginosa (strain
Ruminococcus gnavus
Lactobacillus sakei
Lactobacillus sakei
Lactobacillus sakei
Streptococcus salivarius
Streptococcus pyogenes serotype
Streptococcus salivarius
Streptococcus salivarius
Staphylococcus aureus
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes serotype
Bacillus subtilis (strain 168)
Bacillus subtilis
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Bacillus cereus (strain ATCC 14579/
Bacillus cereus 95/8201
Bacillus cereus 95/8201
Bacillus thuringiensis
Rhizobium leguminosarum bv. trifolii
Streptococcus uberis
Streptococcus uberis
Clostridium perfringens
Micrococcus varians
Streptococcus equi subsp.
zooepidemicus
Myxococcus fulvus
Streptomyces griseoluteus
Streptoverticillium
griseoverticillatum
Carnobacterium sp. (strain UI49)
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. lactis
Streptomyces sp. (strain A647P-2)
Actinoplanes liguriae
Lactobacillus curvatus
Lactobacillus sakei
Streptococcus mutans
Enterococcus mundtii
Lactobacillus sakei
Lactobacillus paracasei
Leuconostoc mesenteroides
Leuconostoc mesenteroides
Bacillus subtilis
Bacillus licheniformis
Lactococcus lactis subsp. lactis
Serratia plymuthica
Halobacterium sp. (strain AS7092)
Bacillus subtilis
Lactobacillus curvatus
Lactobacillus curvatus
Lactobacillus curvatus
Bacillus thuringiensis subsp.
entomocidus
Lactobacillus curvatus
Carnobacterium divergens
Enterococcus sp.
Enterococcus faecium
Paenibacillus polymyxa (Bacillus
polymyxa)
Staphylococcus epidermidis
Enterococcus faecium
Paenibacillus polymyxa (Bacillus
polymyxa)
Bacillus circulans
Paenibacillus polymyxa (Bacillus
polymyxa)
Lactobacillus rhamnosus
Bacillus licheniformis (strain DSM
Lactobacillus plantarum
Lactobacillus acidophilus
Enterococcus faecalis
Candida activity
Anabaena variabilis ATCC 29413
Nostoc sp 7120
Nostoc azollae 0708
Acaryochloris marina MBIC11017
Cyanothece PCC 8801
Cyanothece PCC 8802
Cyanothece PCC 7424
Cyanothece ATCC 51142
Cyanothece CCY0110
Microcoleus chthonoplastes PCC
Nostoc sp 7120
Anabaena variabilis ATCC 29413
Nodularia spumigena CCY9414
Nostoc azollae 0708
Synechococcus PCC 7335
Prochlorococcus marinus MIT 9303
Lactobacillus curvatus
Bacillus thuringiensis
Lactobacillus curvatus L442
Carnobacterium divergens
Microbispora sp. (strain 107891)
Enterococcus sp.
Enterococcus faecium
Paenibacillus polymyxa (Bacillus
polymyxa)
Staphylococcus epidermidis
Enterococcus faecium
Paenibacillus polymyxa (Bacillus
polymyxa)
Bacillus circulans
Paenibacillus polymyxa (Bacillus
polymyxa)
Lactobacillus rhamnosus
Bacillus licheniformis (strain ATCC
Pseudomonas aeruginosa (strain
Lactobacillus plantarum
Lactococcus lactis subsp. lactis
Lactobacillus acidophilus
Lactobacillus salivarius cp400
For example, in some embodiments, an anti-fungal activity (such as anti-yeast activity) is desired. A number of bacteriocins with anti-fungal activity have been identified. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against yeast strains (Adetunji and Olaoye (2013) Malaysian Journal of Microbiology 9: 130-13, hereby incorporated by reference), an Enterococcus faecalis peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO: 727) has been shown to have neutralizing activity against Candida species {see Shekh and Roy (2012) BMC Microbiology 12: 132, hereby incorporated by reference in its entirety), and bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (Shalani and Srivastava (2008) The Internet Journal of Microbiology. Volume 5 Number 2, hereby incorporated by reference). By way of example, botrycidin AJ1316 (Zuber, P et al. (1993) Peptide Antibiotics. In Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics ed Sonenshein et al., pp. 897-916, American Society for Microbiology, hereby incorporated by reference) and alirin BI (Shenin et al. (1995) Antibiot Khimioter 50: 3-7, hereby incorporated by reference) from B. subtilis have been shown to have antifungal activities. As such, in some embodiments, for example embodiments in which neutralization of a fungal microbial organism is desired, a bacteriocin comprises at least one of botrycidin AJ1316 or alirin B 1.
For example, in some embodiments, bacteriocin activity in a culture of cyanobacteria is desirable. In some embodiments, bacteriocins are provided to neutralize cyanobacteria. In some embodiments, bacteriocins are provided to neutralize invading microbial organisms typically found in a cyanobacteria culture environment. Clusters of conserved bacteriocin polypetides have been identified in a wide variety of cyanobacteria species. For example, at least 145 putative bacteriocin gene clusters have been identified in at least 43 cyanobacteria species, as reported in Wang et al. (2011), Genome Mining Demonstrates the Widespread Occurrence of Gene Clusters Encoding Bacteriocins in Cyanobacteria. PLoS ONE 6(7): e22384, hereby incorporated by reference in its entirety. Exemplary cyanobacteria bacteriocins are shown in Table 5 as SEQ ID NO's 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, and 685.
Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs, and read in view of this disclosure.
As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.
As used herein, a “regulator” or “transcriptional regulator” is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.
The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable markers may be dominant or recessive or bidirectional.
As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.
The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.
The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5′ leader sequence, a coding region and a 3′-nontranslated sequence (3′-end) e.g. comprising a polyadenylation- and/or transcription termination site.
“Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.
In amino acid sequences as described herein, amino acids or “residues” are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to the person skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non-proteinogenic amino acid such as D-amino acids and modified amino acids formed by post-translational modifications, and also any non-natural amino acid, as described herein.
The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO's or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO's. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004.
“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).
A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty=10 (nucleotide sequences)/10 (proteins) and gap extension penalty=0.5 (nucleotide sequences)/0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov/.
Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions.
As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.
Alternative conservative amino acid residue substitution classes:
Alternative physical and functional classifications of amino acid residues:
As used herein, “microbial organism”, “microorganism”, “microbial cell” or “microbial host” and variations of these root terms (such as pluralizations and the like) have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including any naturally-occurring species or synthetic or fully synthetic prokaryotic or eukaryotic unicellular organism. Thus, this expression can refer to cells of any of the three domains Bacteria, Archaea and Eukarya. Exemplary microorganisms that can be used in accordance with embodiments herein include, but are not limited to, bacteria, yeast, filamentous fungi, and algae, for example photosynthetic microalgae. Furthermore, fully synthetic microorganism genomes can be synthesized and transplanted into single microbial cells, to produce synthetic microorganisms capable of continuous self-replication (see Gibson et al. (2010), “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome,” Science 329: 52-56, which is incorporated herein by reference). As such, in some embodiments, the microorganism is fully synthetic. A desired combination of genetic elements, including elements that regulate gene expression, and elements encoding gene products (for example immunity modulators, poison, antidote, and industrially useful molecules also called product of interest) can be assembled on a desired chassis into a partially or fully synthetic microorganism. Description of genetically engineered microbial organisms for industrial applications can also be found in Wright, et al. (2013) “Building-in biosafety for synthetic biology” Microbiology 159: 1221-1235, incorporated herein by reference.
A variety of bacterial species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic bacteria based on a “chassis” of a known species can be provided. Exemplary bacteria with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to, Bacillus species (for example Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis), Paenibacillus species, Streptomyces species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species, Pseudomonas species, Lactobacillus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus, Pseudomonas stutzeri, Clostridium thermocellus, and Escherichia coli. A variety of yeast species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic yeast based on a “chassis” of a known species can be provided. Exemplary yeast with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to Saccharomyces species (for example, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (for example, Candida utilis, Candida krusei), Schizosaccharomyces species (for example Schizosaccharomyces pombe, Schizosaccharomyces japonicus), Pichia or Hansenula species (for example, Pichia pastoris or Hansenula polymorpha) species, and Brettanomyces species (for example, Brettanomyces claussenii).
A variety of algae species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic algae based on a “chassis” of a known species can be created. In some embodiments, the algae comprises, consists essentially of, or consists of photosynthetic microalgae. Exemplary algae species that can be useful for biofuels, and can be used in accordance with embodiments herein, include Botryococcus braunii, Chlorella species, Dunaliella tertiolecta, Gracilaria species, Pleurochrysis carterae, and Sargassum species. Additionally, many algaes can be useful for food products, fertilizer products, waste neutralization, environmental remediation, and carbohydrate manufacturing (for example, biofuels).
A variety of filamentous fungal species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic filamentous fungi based on a “chassis” of a known species can be provided. Exemplary filamentous fungi with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Peniciffium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.
Species include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of” meaning that a peptide or peptidomimetic, a polypeptide, a culture medium, a microbial organism or a composition as described herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, the verb “to consist” may be replaced by “to consist essentially of” meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention.
Reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
As used herein, with “at least” a particular value means that particular value or more. For example, “at least 2” is understood to be the same as “2 or more” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, . . . , etc.
The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.
As used herein, the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
Each embodiment as identified herein may be combined together unless otherwise indicated.
All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.
The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.
(A) Scheme of genomic organization and transcriptional dependencies (dashed arrows) between competence activation (comX) and bacteriocins production (blpK, slvX, . . . ) in S. salivarius. Promoters are depicted with broken arrows. Regulators and the ComS pheromone are stained according to their encoding genes. The ComS precursor is produced (curled arrow) as an intracellular precursor (square) before secretion, maturation and import as an active pheromone (ellipses). The newly described two-Rgg system is shaded and the T arrow pinpoints the inhibitory role of SarF on ScuR.
(B, C, D and E) Maximum luciferase activity/OD600 ratio (RLU/OD; logarithmic scale) of various competence or bacteriocin production-involved promoters fused to a luxAB reporter system in WT or overexpressing backgrounds. (B) Promoter activation of genes upon sComS addition (full bars) vs mock condition (striped bars) in WT (light grey bars) or scuR overexpression mutant (scuR++; dark grey bars). (C) Activity of sptA, comS and slvX promoters in WT strain, and scuR++ or sarF (sarF++) overexpression mutants. (D) Activity of sptA and comS promoters in WT and scuR++ mutant deleted or not for comR gene. (E) Activity of sptA and comS promoters in overexpression of scuR or sarF in a scuR locus (ΔscuR-sarF) deletion background. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(F) Maximum luciferase activity/OD600 ratio (RLU/OD) of comS, blpK and slvX promoters fused to a luxAB reporter system in WT (open bars) or ΔscuR-sarF (grey bars) strain activated with sComS. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(A) Cartoon portraying the rational strategy for the peptide randomization-based screen. A library of randomized small genes under inducible promoter controls (Pxyl) is transformed in a reporter strain in which the chloramphenicol resistance gene (cat) is translationally fused to sptA promoter. In absence of xylose or upon xylose induction of irrelevant peptides (square, hexagon and star), sptA promoter is maintained OFF and does not initiate cat transcription, causing cell sensitivity (CmS) on chloramphenicol-supplemented media. The xylose-driven intracellular production of a cognate peptide (ellipse) promotes chloramphenicol resistance (CmR), through PsptA activation by ScuR/SarF (dashed arrow).
(B) Activity of sptA promoter in WT strains and various mutants expressing intracellularly activating peptides (cartoon) in medium supplemented with xylose (0.1% or 1%; grey bars) vs mock conditions (open bars). The BM 1 clone is an irrelevant peptide (negative control). Magnitude is expressed in percentage compared to the WT PsptA-luxAB reporter strain (Relative maximal luciferase activity). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(C) Weighted consensus sequence for the suite of 22 activating peptides identified in the randomization-based screen. Randomized residues are crowned with a horizontal black bar while the non-variable amino acids are grey-coloured. The Bits represent the relative frequency of residues. Information content is plotted as a function of residues position and reckoned from the N-terminus (1 to 12) or the C-terminus (−1 to −12). The sequence logo image was generated using the WebLogo application (accessible on the world wide web at weblogo.berkeley.edu/logo.cgi).
(D) Promoter activity of the sptA gene in response to the BI7 encoded peptide in various rgg mutant backgrounds. Media were supplemented with 0.1% xylose (open bars) or water (grey bars). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(A) Cartoon depicting the ScuR/SarF-mediated activation of PsptA upon addition of exogenous synthetic peptides.
(B) Fold increase in maximal PsptA activity upon addition of representative synthetic peptide (0.01 or 1 μM) vs mock conditions. Peptide sequences are correlated to peptide name and compared to the consensus motive (SEQ ID NO: 728) (open box). sBH6: SEQ ID NO: 729; sBI7: SEQ ID NO: 730; sBI10: SEQ ID NO: 731; sBJ1: SEQ ID NO: 732; sBK1: SEQ ID NO: 733; sBK3: SEQ ID NO: 734; sBK4: SEQ ID NO: 735; sBK8: SEQ ID NO: 736; sBO2: SEQ ID NO: 737. The crucial W and G residues are highlighted with grey boxes.
(C) Maximal activity of PsptA exposed to sBI7 WT and mutant peptides (1 nM).
(D) Dose response dot plot of sptA, slvX and comS promoter activity upon sBI7 induction at various concentrations (nM). Promoters were tested in WT strain and, ΔscuR or ΔsarF deletants.
(B, C and D) Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(E) Maximal activity of PsptA (RLU/OD; logarithmic scale) exposed to WT and mutant sBI7 peptides (1 μM). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(F) Activity of sptA, comS and slvX promoters (absolute maximal luciferase activity; ALU) in ΔscuR-sarF challenged with sBI7 synthetic peptide (grey bars) vs mock conditions (open bars). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(G) Maximum luciferase activity/OD600 ratio (RLU/OD; logarithmic scale) of comS, slvX and sptA promoters fused to a luxAB reporter system in WT (open bars) or ΔcomR (grey bars) strain activated with the sBI7 synthetic peptide. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(A) Mobility shift assays of comX, comS, slvX and sptA promoter probes conducted with purified Rgg paralogs and decreasing concentrations of their cognate peptide (grey triangles; 2:2 dilutions from 20 μM). Probes are 30 bp (or 40 bp for PsptA), were Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangles showcase ternary complexes (peptide-Rgg-DNA).
(B) Nucleotide alignment of promoters of comX (SEQ ID NO: 738), comS (SEQ ID NO: 739), slvX (SEQ ID NO: 740) and sptA (SEQ ID NO: 741). The palindromic stretches (inverted arrows) and the sigma-bound DNA sequence (−10 boxes) are shaded in grey. Boxed nucleotides highlight the potential mismatches in the hairpin structure of PcomX or PsptA that were substituted to restore a genuine dyad symmetry sequence (see
(C and D) Fold increase in maximal activity of WT and mutated promoters of sptA (C) or comX (D) exposed to sBI7 or sComS (1 μM). Nucleotides substitutions and insertions are disclosed in
(E) Mobility shift assays of mutated comX promoter probes conducted with a unique concentration of ScuR or SarF (grey boxes; 4 μM) and decreasing concentrations of sBI7 peptide (grey triangles; 2:2 dilutions from 20 μM). Open triangles showcase ternary complexes (peptide-Rgg-DNA).
(F) Mobility shift assays of comS, slvX and sptA promoter probes conducted with purified ScuR and decreasing concentrations of the non-cognate sComS peptide (grey triangles; 2:2 dilutions from 20 μM). Probes are 30 bp (or 40 bp for PsptA), were Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangle showcases binary complexes (ScuR-DNA).
(G) Mobility shift assays of slvX and sptA promoter probes conducted with sBI7 peptide and decreasing concentrations of purified ScuR (grey triangles; 2:2 dilutions from 8 μM). Probes are 30 bp (PslvX) or 40 bp (PsptA), were Cy3-conjugated and used at 40 ng. Peptide concentration remains constant (grey boxes; 1 μM). Open triangles showcase ternary complexes (sBI7-ScuR-DNA).
(H) Nucleotide alignment of bacteriocin-related gene promoters. PslvX: SEQ ID NO: 740; P00176: SEQ ID NO: 742; P01584: SEQ ID NO: 743; PblpK: SEQ ID NO: 744; PslvV: SEQ ID NO: 745; PblpG: SEQ ID NO: 746; PslvW: SEQ ID NO: 747; PslvY: SEQ ID NO: 748. The palindromic stretches (inverted arrows) and the sigma-bound DNA sequence (−10 boxes) are shaded in grey. The characteristic T-rich region is grey font.
(A and B) Bacteriocin inhibition assay of S. salivarius WT and mutant derivatives. Indicator strains (L. lactis) were embedded in the top soft agar layer, while sBI7 was supplemented into the bottom agar layer as required. Producer strains were spotted on top of the two agar layers. (A) Killing properties of scuR or sarF overexpression mutants compared WT. (B) Effect of sBI7 addition (1 μM) on WT strain and scuR/sarF various mutants. scuR++ and bacteriocin null mutant (Δslv5) were used as positive and negative control, respectively.
(C) Bacteriocin inhibition assay of S. salivarius WT and scuR/sarF mutant derivatives. Indicator strains (L. lactis) were embedded in the top soft agar layer, while sBI7 was supplemented or not into the bottom agar layer as stated. Producer strains were spotted on top of the two agar layers. scuR++ and bacteriocin null mutant (Δslv5) were used as positive and negative control, respectively.
(A) Conservation of ScuR locus, and BIpRH system across S. salivarius. Functional BIpRH pair, ScuR, SptA, SptB, and SarF were sought for homologs in various S. salivarius strains. The phylogenetic tree (100 bootstrap replicates) was adapted from (Yu et al., 2015). An empty box means that no functional ortholog was found in the species genome. Scale bar: 0.01 substitution per site.
(B) Figurative illustration of RRNPPs vs two-component systems (TCSs) drift toward competence and predation regulation in paradigmatic streptococci (S. pneumoniae, S. mutans and S. salivarius).
Maximal activity of salivaricin promoters (RLU/OD; logarithmic scale) exposed to sBI7 (WT) (SEQ ID NO: 36) and mutant peptides (1 μM). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
Maximal activity of PcomX exposed to the sComS or sBI7 peptide (1 μM) (SEQ ID NO: 36) in WT strain and ΔscuR and/or ΔsarF mutants. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
Maximal activity of PcomX and ComX-dependent promoters exposed to the sComS (light grey) or sBI7 peptide (SEQ ID NO: 36) (dark grey) (1 μM) in comparison to basal activity (open box). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(A) Maximal activity of PsptA exposed to LAFWDSLG (SEQ ID NO: 749), LAFWDSLGLLL (SEQ ID NO: 750) or the peptide sBI7 (SEQ ID NO: 36) (1 μM) in comparison to basal activity. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.
(B) Mobility shift assays of the sptA promoter probe conducted with purified ScuR and decreasing concentrations of the peptides LAFWDSLG (SEQ ID NO: 749) or LAFWDSLGLLL (SEQ ID NO: 750) (grey triangles; 2:2 dilutions from 20 μM). The probe is 40 bp, Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangle showcases single or binary complexes (ScuR-DNA).
Bacterial strains, plasmids and oligonucleotides used in the Examples are listed and described in the tables below. Streptococcus salivarius HSISS4 and derivatives were grown at 37° C. without shaking in M17 (Difco Laboratories, Detroit, Mich.) or in CDM (Fontaine et al. Mol Microbiol 2013, 87:1113-1132) supplemented with 1% (w/v) glucose (M17G, CDMG, respectively). Escherichia coli TOP10 (Invitrogen) were cultivated with shaking at 37° C. in LB. Electrotransformation of E. coli was performed as previously described (Mignolet et al. Elife 2016, 5:e18647). Lactococcus lactis was grown in M17 broth with 1% glucose at 30° C. without shaking. We added 1.5% (w/v) agar into M17 and LB plates, and bacteriocin inhibition tests were assayed on M17 plates containing 0.2% agar. We added D-xylose (0.1 or 1%; w/v), ampicillin (250 μg/ml), spectinomycin (200 μg/ml), chloramphenicol (5 μg/ml; except if otherwise stated) or erythromycin (10 μg/ml), 5-FOA (1 mg/ml) (Melford Laboratories), as required. Synthetic peptides and sComS (purity of 95%; 1 μM, except if otherwise stated) were supplied by Peptide 2.0 Inc. (Chantilly, Va., USA) and resuspended in DMSO. Solid plates inoculated with streptococci cells were incubated anaerobically (BBL GasPak systems, Becton Dickinson, Franklin lakes, N.J.) at 37° C.
Escherichia coli
Streptococcus salivarius
Lactococcus lactis subsp. lactis
S. salivarius, encoding the Cre recombinase; eryR
To generate the two DNA libraries encoding randomized sequence of small peptides, we performed overlapping PCRs to graft fragments encompassing the follow features: (1) a 5′ recombination arm (for the ectopic tRNAser locus); (2) the xylR gene that codes for the xylose responsive regulator; (3) either Pxyl1 (library I) or Pxyl2 (library II) translationally-fused to a 12 codon long gene, for which the last 7 are randomized; (4) the spec gene; (5) a 3′ recombination arm (for the ectopic tRNAser locus). To obtain the randomized DNA stretch, we used a 78 nucleotide long primer degenerated on 21 contiguous positions. Next, we transformed these two libraries in strains containing the sptA promoter translationally-fused to the cat gene (chloramphenicol resistance) in which the associated spec gene was excised by the previously described cre-lox method. The initial background of these strains were either a comR-overexpressing (Pxyl1-comR) or a salivaricin-deprived (Δslv5) strain. We plated transformed cells on solid medium supplemented with xylose (either 0.1 or 1%), chloramphenicol (2 mg/ml) and spectinomycin (200 mg/ml) and grew overnight. We restreaked single colonies on fresh chloramphenicol and spectinomycin solid medium supplemented or not with xylose. We finally collected clones that displayed an increased in growth on xylose vs non xylose medium (except for the clone BM1 that we used as a negative control).
All double-stranded DNA fragments (30 or 40 bp) were obtained from annealing of single-stranded Cy3-labelled (at 5′ end) and unlabeled oligonucleotides. Primers used are listed in Table 3. Typically, a gel shift reaction (20 μl) was performed in a binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1 mg ml-1 BSA) and contained 150 ng labelled probe and 4 mM StrepTag proteins. When necessary, 8 mM of ComS peptides (unless otherwise stated) are added. The reaction is incubated at 37° C. for 10 min prior to loading of the samples on a native TBE 5% gel. The gel is next subjected to 80 V for approximately 1 h in TBE buffer. DNA complexes were detected by fluorescence on the Ettan DIGE Imager with bandpass excitation filters (nm): 540/25 (Cy3) or 635/30 (Cy5) and bandpass emission filters: 595/25 (Cy3) or 680/30 (Cy5) (GE Healthcare, Waukesha, Wis.).
The spot-on lawn (multilayer) detection method was performed as followed: 10 μl of overnight cultures of producer strains were diluted in fresh M17G medium and grown to reach mid-log phase (OD600=˜0.5). In parallel, we casted plates with a bottom feeding layer (M17G 1.5% agar) supplemented with a synthetic peptide where required. Next, we mixed 100 μl of an overnight culture of Lactococcus lactis IL1403 (indicator strain) in pre-warmed soft M17G medium (0.3% agar) and casted it as a top layer. Finally, we incubated mid-log phase cultures for 30 minutes with the corresponding synthetic peptides and spotted 3 μl of the producer strains on the top layer. Plates were incubated overnight before analysis of the inhibition zones surrounding the producer colonies.
To induce competence, overnight CDMG precultures were diluted at a final OD600 of 0.05 in 300 μl (10 ml concerning the randomized peptide screen) of fresh CDMG and incubated 75 min at 37° C. Then, we added the pheromone sComS as well as DNA (overlapping PCRs or plasmids) and let the cells recover for 3 h at 37° C. before plating on M17G agar supplemented with antibiotics where required. Null-mutants were constructed by exchanging (double homologous recombination) the coding sequences (CDS) of target genes (sequence between start and stop codons) for either chloramphenicol or erythromycin resistance cassette. If stated, mutants were cleaned for the lox site-flanked resistance cassette, as previously described (Fontaine et al. Mol Microbiol 2010; 87:1113-1132). In case of deletion of multiple CDSs, the region between the start codon of the first CDS and the stop codon of the last CDS was deleted. Integration of the antibiotic resistance cassette at the right location was subsequently checked by PCR. The promoters of sptA genes was fused to the luxAB reporter genes and inserted with a chloramphenicol resistance cassette at the permissive tRNA threonine locus (HSISS4_r00061) by double homologous recombination. In case of ΔscuR and ΔsarF in-frame deletion, we used the two-step selection/counter-selection strategy previously described (Mignolet et al. Cell Rep 2018, 22:1627-1638). We transformed the wild-type strain with an overlapping PCR product composed of 4 fragments: (I) the upstream region of scuR or sarF genes, (II) the downstream region of scuR or sarF genes, (III) a cassette that includes the erythromycin resistance gene (erm) and a gene encoding the orotate transporter oroP, and finally (IV) the downstream region of scuR or sarF genes. We selected a first event of double recombination on medium supplemented with erythromycin. Next, we selected an intramolecular recombination between region (I) and (IV) that excises the erm-oroP cassette by growing cells on M17G supplemented with the toxic 5-fluoro-orotic acid (5-FOA) compound. In absence of oroP, 5-FOA is not able to cross the membrane and penetrate the cytoplasm where it is deleteriously incorporated in the nucleotide metabolic pathway (Overkamp et al., 2013). At final, we engineered an in-frame deletion mutant of scuR or sarF in which the first seven codons were fused to the last six codons without any cassette scar (see below for detailed cloning method).
The PCR-amplified scuR-StrepTag and gene sarF-StrepTag were cloned into the pBAD-comR-ST vector (see supplemental information). The ComR-StrepTag, ScuR-StrepTag and SarF-StrepTag recombinant proteins were overproduced in E. coli and purified as previously described (Fontaine et al., 2013) in standard native conditions on Strep-Tactin agarose beads (IBA).
Overnight precultures were diluted at a final OD600 of 0.05. A volume of 300 μl of culture samples was incubated in the wells of a sterile covered white microplate with a transparent bottom (Greiner, Alphen a/d Rijn, The Netherlands) for 75 min at 37° C. and then supplemented with synthetic peptides (1 μM, except if otherwise stated) or DMSO, and xylose where required. Growth (OD600) and luciferase (Lux) activity (expressed in relative light units) was monitored at 10 min intervals during 24 h in a multi-wells plate reader (Hidex Sense, Hidex, Turku, Finland) as previously described (Fontaine et al., 2013).
S. salivarius WT, ΔscuR, ΔsarF, scuR++ or ΔsarF-ST++ strains were pre-cultured overnight in CDMG at 37° C. They were resuspended in 50 ml of fresh pre-warmed CDMG to a final OD600 of 0.05 and grown for approximately 2 h 30 min (OD600=0.3) at 37° C. Cells were harvested by centrifugation (10 min; 4,050×g), the supernatant were discarded and the cell pellets were frozen with liquid nitrogen. Finally, RNA was extracted using the RiboPure bacteria kit (Ambion-Life Technologies) and the protocol provided by the manufacturer, with protocol changes to cell lysis and RNA precipitation. For lysis, cells were resuspended in RNAwiz buffer (Ambion-Life Technologies) supplemented with Zirconia beads and shaked for 40 sec (4 times) in a fastPrep homogenizer device (MP biomedicals). For RNA precipitation, a 1.25-ethanol volume (instead of 0.5) was added to partially purified RNAs. Total RNA was checked for quality on a RNA Nano chip (Agilent technologies) and concentration was measured using Ribogreen assay (Life technologies). rRNA depletion was performed on 2 μg total RNA with the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina) according to manufacturer's instructions. Total stranded mRNA libraries were prepped with the NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). Library PCR was executed for 15 cycles. Quality of the libraries was evaluated with the use of a High sensitivity DNA chip (Agilent technologies) and concentrations were determined through qPCR according to Illumina protocol. Libraries were sequenced on a NextSeq 500 high-throughput run with 76 bp single reads. 2.3 pM of the library was loaded on the flowcell with a Phix spike-in of 5%. Sequenced mRNAs generated several million reads that were mapped on the WT S. salivarius chromosome and processed with both bowties V0.12.9 (http://bowtie-bio.sourceforge.net/bowtie2) and samtools V0.1.18 (http://samtools.sourceforge.net/) algorithms to yield BAM files containing the read coordinates. We imported these files into SeqMonk V0.23.0 (www.bioinformatics.babraham.ac.uk/projects/) to assess the total number of reads for each coding sequence (CDS). The dataset was exported into an excel file for further analyses. First, the dataset was standardized to CDS-mapped reads per million overall reads. Then, we estimated a ratio of CDS-mapped reads in mutants vs WT. All RNAseq data was deposited in the GEO database under accession number GSE120640.
All DNA fragments were amplified by PCR using the Phusion high fidelity polymerase (www.thermoscientificbio.com/) following a protocol as recommended by the manufacturer. Overlapping PCR products were transferred in competence-induced HSISS4 derivatives (Mignolet at al. Genome Announc 2016, 4:e01637-01615). cat, erm, spec, erm-oroP, Pxyl1, Pxyl2, and luxAB-cat cassettes were amplified from pNZ5319, pGIUD0855ery, pJUDspecmut1-gfp+ter, pSEUDO-Pusp45-sfgfp(Bs), pZX9, pZX10 and pJIMcat, respectively. comX and sptA mutated promoter were amplified from the WT comX and sptA luxAB-fused promoter strain, respectively. The sarF-ST allele was amplified from pBAD-sarF-ST. The full Pxyl1-comR-spec at tRNAsSer locus was amplified in one block from the strain tRNASer::Pxyl1-comR-spec (Mignolet et al. Cell Rep 2018, 22:1627-1638). All the constructed plasmids were sequence-verified.
pBAD-scuR-ST. The scuR-coding sequence was PCR amplified using the rggD_NcoI SS and rggD_MunI primers. This scuR fragment was digested with NcoI/MunI and cloned into NcoI/EcoRI-digested pBAD-comR-ST (Mignolet et al. Cell Rep 2018, 22:1627-1638).
pBAD-sarF-ST. The sarF-coding sequence was PCR amplified using the rggC_NcoI SS and rggC_RI SS primers. This sarF fragment was digested with NcoI/EcoRI and cloned into NcoI/EcoRI-digested pBAD-comR-ST (Mignolet et al. Cell Rep 2018, 22:1627-1638).
The bacteriocin short-circuitry imposed by ComR in the S. salivarius species is startling and suggests a positive selection for species-specific strategies that participate in niche adaptation. Interestingly, the S. salivarius HSISS4 genome encodes five RRNPP transcriptional factors, including ComR. Besides it, the two regulators ScuR (HSISS4_01166; stands for salivaricins-competence uncoupling regulator) and SarF (HSISS4_01169; ScuR-associated Rgg factor) share a high level of similarity with ComR (64 and 63%, respectively). The residues involved in HTH sequestration and homodimerization of ComR are well-conserved in both ScuR and SarF, suggesting that they could display a similar mode of activation. Moreover, the paralogous ScuR and SarF proteins are highly identical (similarity of 91%). Strikingly, residue divergences are nearly all concentrated in only 3 amino-acids stretches, one of which overlaps the α-helices 13 and 14 that form a part of the peptide recognition pocket. This indicates that the two proteins are likely to homodimerize and could accommodate specific peptides. On the chromosome, the scuR and sarF genes are located in a unique locus and separated by two genes that code for two predicted subunits of an ABC transporter, SptA and SptB (for ScuR-promoted transporter A and B, respectively) (
Due to the huge conservation between ComR, ScuR and SarF, especially in the DNA binding domain, we questioned whether the two uncharacterized paralogs are capable to control competence and predation as well. Hence, we extracted mRNA of wild-type (WT) and engineered in-frame deletion mutants (ΔscuR and ΔsarF) and carried out a deep sequencing (RNAseq). With no hint about the genuine activating pheromones, we included in our high-throughput transcriptomic analyses overexpression mutants (scuR++ and sarF-ST++), in order to exacerbate the regulation phenotype. Indeed, a strong overproduction of ComR was reported to be sufficient for activation of its target promoters, even in absence of ComS (from endogenous production or synthetic peptide addition). Both deletion mutants did not dramatically affect the transcriptome compared to the WT strain, meaning that the function of ScuR and SarF is barely noticeable during standard growth conditions. However, the SarF loss slightly increased scuR expression, while the differential sptA and sptB mRNA level almost reached the arbitrary 5-fold induction cut-off, suggesting that SarF could be a repressor/antagonist of the ScuR-SptAB system. In contrast, the strong overexpression of scuR (28-fold) elicited a tremendous activation of the sptA-sptB operon (about 2000-fold). Furthermore, a second cluster of genes, all located inside salivaricin loci, was robustly influenced, even if with a lower magnitude of activation (ranging from 35- to 140-fold). Surprisingly, comX mRNA levels remained approximately stable in all mutants.
Altogether, these results imply that the ComR, ScuR and SarF paralogs might shape overlapping but dedicated regulatory networks.
In order to validate our transcript profile analyses, we performed promoter-probe assays, as previously described for ComR. We first expanded our collection of luciferase reporter strains (composed of comS, comX and bacteriocin gene promoters) to include and monitor the sptA promoter (PsptA) and, next transformed all of them with the scuR overexpression cassette. Finally, we measured promoter activity in presence or absence of sComS (synthetic peptide) during cell growth (
Considering that the effects of transcriptional regulators could be indirect, we were prompted to inactivate one Rgg by gene deletion in our reporter strains and test the residual activity of the others. We discovered that ScuR still controls both PsptA and PcomS in comR (
Taken together, our results suggest that the 3 transcriptional factors have only partly redundant functions, with regulatory network specificities, presumably to ensure a broader diversity of cellular response to environment stresses. ScuR and SarF, but not ComR, control the sptAB operon, while ScuR alone has a ComR-independent extra regulatory role on bacteriocin production. Even though ScuR raises ComS production, this regulator does not act on comX promoter and is likely to disconnect the competence-predation coupling compelled by ComR.
Typically, the major challenge to address the transduction mechanism of cell-cell communication sensor is the identification of the ligand(s) or the perceived signal(s). As inspection of the genomic neighborhood did not reveal any small encoded peptide in the vicinity of scuR-sarF locus, we decided to conduct an empirical screen to unearth peptides able to activate the ScuR-SarF system (
In order to discard clones with secondary mutations for which the survival phenotype was not related to the peptide nature, we amplified for each clone the full locus that encodes the small peptide and backcrossed it into WT or (Δslv5) backgrounds. We then confirmed on solid media that chloramphenicol resistance qualitatively increased upon xylose addition. We used the same PCR products to transform a strain bearing the PsptA-luxAB report fusion and quantitatively estimate the influence of peptide production. Again, we noticed that xylose addition potentiated the promoter activity with values ranging from 5 to 100 fold, while it has no effect on BM1 (negative control) and WT strains (
We aligned the 22 unique peptide sequences to elicit common chemical properties (
We next checked whether we could, akin to ComS toward ComR, activate the system with synthetic peptides (
To refine our view of the 2 Rgg-systems, PsptA, PcomS and PslvX were challenged with increasing amount of sBI7 at low concentration in WT, ΔscuR or ΔsarF strains (
Next, we carried out in vitro mobility shift assays to assess the direct interaction between proteins and promoter probes in absence or presence of decreasing concentration of synthetic peptides. We included a PcomX probe as a negative control. Anew, we corroborated our promoter activity data at the magnitude and protein-peptide/DNA specificity level (
The topology of ScuR, SarF or ComR responsive promoters appears extremely similar (
The lower reactivity of salivaricin promoters toward ScuR (vs ComR) pheromone incited us to investigate the phenotypical output at the bacteriocin production level. Thence, we performed a standard bacteriocin test on soft overlay and showed that the scuR++ (but not sarF-ST++) overexpression mutant is able to produce a comA-dependent inhibitory halo in bacteriocin tests (
ScuR tends to compensate for the loss/lack of a functional BIpRH system, as they usually do not co-exist in the same strain (
All our phenotypical and molecular data coincide to conclude that ScuR is strictly devoted for predation in contrast to competence. Although highly similar to PcomS and PslvX at primary sequence level, comX promoter cannot be occupied (
Confirming the importance of the ultra-conserved tryptophane residue (see
It was also confirmed that ScuR and SarF have no effect on the sComS-mediated competence entry (
acalculated as the ratio of transformants (chloramphenicol-resistant CFU) to the total CFU count per 0.1 ug of linear DNA. Transformation frequencies are expressed as the arithemtic mean of three independent experiments. Geometric means ± standard deviations are provided. ND: not detected (<1.0E−08), NA: not applicable.
To evaluate whether the synthetic pheromones could tolerate amino acid addition at the C-terminus, we tested the close-to-consensus petide LAFWDSLG (SEQ ID NO: 749) as well as the peptide LAFWDSLGLLL (SEQ ID NO: 750) that harbors a triple leucine extension at the C-terminus. Strikingly, both peptides were able to induce the sptA promoter in vivo. However, the longer peptide was much more active at 1 μM concentration than the shorter one, with an induction magnitude comparable to the peptide sBI7 (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 18207694.3 | Nov 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/082242 | 11/22/2019 | WO | 00 |
