WO2007020617A2 - Produit - Google Patents

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WO2007020617A2
WO2007020617A2 PCT/IE2006/000088 IE2006000088W WO2007020617A2 WO 2007020617 A2 WO2007020617 A2 WO 2007020617A2 IE 2006000088 W IE2006000088 W IE 2006000088W WO 2007020617 A2 WO2007020617 A2 WO 2007020617A2
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seq
nucleic acid
fragment
acid sequence
variant
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PCT/IE2006/000088
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WO2007020617A3 (fr
WO2007020617A9 (fr
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Paul W. O'toole
Gerald F. Fitzgerald
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University College Cork-National University Of Ireland, Cork
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to the identification and isolation of genes in the Lactobacillus salivarius genome involved in probiotic activity or function, and a novel
  • WO98/35014 describes strains of Lactobacillus salivarius isolated from resected and washed human gastrointestinal tract which inhibit a broad range of Gram positive and Gram negative microorganisms and which secretes a product having antimicrobial activity into a cell-free supernatant.
  • WO00/41707 describes the use of Lactobacillus salivarius in the prophylaxis or treatment of undesirable inflammatory activity, especially gastrointestinal activity such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis or Crohn's disease.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • ulcerative colitis Crohn's disease
  • the inflammatory activity may also be due to cancer.
  • the strain therefore has use in the prophylaxis or treatment of a number of disease states including gastrointestinal inflammatory activity such as pouchitis, post infection colitis, diarrhoeal disease associated by Clostridium difficile or with Rotovirus, post infective diarrhoeal disease, inflammatory activity due to
  • Lactobacillus salivarius subsp salivarius strain UCCl 18 has demonstrated colonisation ability and therapeutic effects in human subjects [1] . It has also been shown to reduce arthritis, inflammation and tumour development in mice. [2].
  • the invention is directed towards a better understanding of the mechanisms involved in their activity.
  • the invention also provides a polynucleotide comprising a nucleic acid sequence of SEQ ID No. 1 or a variant or fragment thereof isolated from a probiotic bacterium.
  • the invention also provides a polynucleotide comprising a nucleic acid sequence of SEQ ID No. 1 or a variant or fragment thereof isolated from Lactobacillus.
  • the polynucleotide is isolated from Lactobacillus salivarius subsp salivarius. Most preferably the polynucleotide is isolated from Lactobacillus salivarius subsp salivarius UCCl 18 [NCIMB 40829].
  • the polynucleotide comprises a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence of SEQ ID No. 1. In one embodiment of the invention the polynucleotide comprises at least 30 contiguous nucleic acids of SEQ ID No. 1.
  • the invention further provides an isolated polynucleotide comprising a nucleic acid sequence selected from any one or more of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, 10 and SEQ ID No. 11 or a variant or fragment thereof or a sequence complementary thereto.
  • the polynucleotide encodes a gene whose function is essential to probiotic activity or function.
  • the isolated polynucleotide encodes a gene involved in any one or more of sugar utilisation and sugar phosphate metabolism, environmental sensing mechanism, adhesion, bile resistance and amino acid metabolism and/or acid resistance.
  • the invention further provides a polypeptide encoded by a polynucleotide as hereinbefore described.
  • the invention further provides a genetic construct comprising a polynucleotide as hereinbefore described.
  • the invention also provides use of a polynucleotide as hereinbefore described in the selection or production of probiotic bacteria.
  • the invention also provides use of recombinant vector comprising a polynucleotide as hereinbefore described.
  • the invention further provides an isolated polynucleotide comprising a nucleic acid sequence selected from any one or more of SEQ ID No. 14 (LSLOl 52), SEQ ID No. 15 (LSLO 152), SEQ ID No. 16 (LSL0311 ), SEQ ID No. 17 (LSL 1085), SEQ ID No. 18 (LSL1319), SEQ ID No. 19 (LSLl 319), SEQ ID No. 20 (LSL 1335), SEQ ID No. 21 (1832b), SEQ ID No. 22 (LSL0350), SEQ ID No. 23 (LSL0351), SEQ ID No. 24 (LSL0352), SEQ ID No. 25 (LSL0873), SEQ ID No. 26 (LSL 140 Ib), SEQ ID No. 27 (LSLHOIb), SEQ ID No. 28 (LSL 1832c), SEQ ID No. 29 (LSL1832d), SEQ ID No. 30 (LSL_1838) or a variant or fragment thereof.
  • SEQ ID No. 14
  • the invention also provides an isolated polynucleotide wherein the nucleic acid sequence is at least 70% identical to the nucleic acid sequence of any one or more of SEQ ID No. 14 to SEQ ID No. 30.
  • the invention also provides an isolated polynucleotide wherein the fragment comprises at least 30 contiguous nucleic acids to the nucleic acid sequence of any one or more of SEQ ID No. 14 to SEQ ID No. 30.
  • the isolated polynucleotide encodes a polypeptide or protein having probiotic function and/or activity. In another embodiment the polynucleotide encodes a gene involved with intestinal cell adherence properties of Lactobacillus.
  • the invention also provides a polypeptide or protein encoded by a polynucleotide as hereinbefore described.
  • the polypeptide or protein is capable of mediating adherence to epithelial cells and modulating epithelial gene expression to improve gut barrier function.
  • polypeptide or protein is used for the preparation of a medicament for use in the prophylaxis or treatment of undesirable inflammatory activity.
  • the polypeptide or protein may also be used to modulate or alter the metabolism of Lactobacillus.
  • the invention further provides a vaccine or formulation comprising a polypeptide or protein as hereinbefore described.
  • the vaccine or formulation may be prepared using commonly used excipients and/or carriers.
  • genome or genomic sequence is taken to mean the sequence of the chromosome of Lactobacillus salfvarius.
  • plasmid is taken to designate any extrachromosomal piece of DNA contained in the Lactobacillus.
  • variant or fragment thereof is taken to mean a derivative fragment generated by minimal genetic modification including but not restricted to point mutation, rearrangement or in vitro optimisation.
  • Fig. 1 is a Genome atlas of L. salivarius UCCl 18;
  • Fig. 2A shows a pulsed field gel electrophoresis (PFGE) of total genomic DNA of Lactobacillus salivarius UCCl 18 run either digested (+) or undigested (-) with the labelled restriction enzymes.
  • PFGE pulsed field gel electrophoresis
  • the in silico fragment number predictions for the chromosome, megaplasmid, pSF44 and pSF20 are indicated in the accompanying table; This shows that there is complete concordance, within the resolution limits of the gel for smaller fragments;
  • Fig. 2B shows a Southern hybridization of the Lactobacillus salivarius UCCl 18 genomic digests from Fig. 2A, using a gene probe based on abpa+abp ⁇ (encoding bacteriocin ABPl 18 structural peptides) localized in the megaplasmid pMPl 18 identified in strain UCCl 18;
  • Lactobacillus salivarius UCCl 18 adhesion to intestinal epithelial cells (HT29 cell line). Values graphed are the mean of three independent biological replicates, each with technical duplicates. Error bars are standard error of the mean;
  • Fig. 5A & B Impaired epithelial cell adhesion by mutants lacking sortase, or sortase-dependent proteins, in Lactobacillus salivarius UCCl 18.
  • Fig. 6 is a graph showing the functional categorisation of the UCCl 18 genome with the gene content assigned to functional categories;
  • Fig. 7 is a table showing the relatedness of the UCCl 18 to other Lactobacilli.
  • the table shows a summary of organism distribution among the 10 best Smith- Waterman hits for each UCC 118 gene from the four replicons;
  • Fig. 8A is a pulse field gel electrophoresis of Z. salivarius; Fig. 8B is a southern hybridisation corresponding to Fig 8A. Megaplasmids of varying size are found in L. salivarius.
  • Panel A PFGE separation of genomic DNA of 10 Z. salivarius strains.
  • Panel B Corresponding Southern hybridization with the LSL_1739 repA probe. (+) or (-) indicate treatment with Sl nuclease. Arrows to left indicate ⁇ DNA concatamers used as size standard. Prominent bands in Panel A, Sl nuclease treated samples, are chromosomal DNA. In panel B, non-specific hybridization with
  • the estimated size of megaplasmids are: AH4231 , 210 kb; UCC 118, 242 kb; UCC 119, 195 kb; DSM20492, 240 kb; DSM20555, 380 kb; NICMB8816, 180 kb;
  • Fig. 9 is a diagrammatic representation of the molecular organisation of the intact sortase dependent proteins of Z. salivarius UCCl 18 and their homologues.
  • Fig 1OA is a southern hybridisation of a sortase gene deletion mutant
  • Fig. 1OB is a schematic overview of the genome structure of a sortase gene deletion mutant. Verification of the genome structure of a sortase-gene deletion mutant.
  • Panel A Southern hybridization. The fragments expected following digestion are indicated by arrows, and fragments sizes are indicated in kilobase pairs.
  • Panel B Schematic overview. The sortase gene is indicated as a box whereas the probe is indicated as a hatched box
  • Fig. 1 1 shows expression analysis of genes encoding sortase-dependent proteins. Transcription of genes for sortase-dependent proteins investigated by RT-PCR. PCR was performed on cDNA prepared from stationary phase cells grown in MRS broth. Arrows indicate sizes in base pairs. Genes are indicated above the lanes. Gene labels with 5' or 3' suffices indicate expression was tested upstream or downstream of the internal stop codon.
  • Lactobacillus salivarius UCCl 18 was sequenced and analysed using standard methodology.
  • the circular megaplasmid is composed of 242,436 basepairs.
  • the nucleic acid sequence for the megaplasmid is given in SEQ ID No. 1.
  • the megaplasmid has been named pMPl 18.
  • pMPl The megaplasmid plays a major role in the probiotic properties of Lactobacillus salivarius.
  • a plasmid pSF-20 composed of 20,417 basepairs
  • a circular chromosome composed of 1,827,111 basepairs (NCBI database accession no. CP000233)
  • Table 1 shows the content of the UCCl 18 genome
  • the genome sequence of L. salivarius subsp. salivarius strain UCCl 18 consists of 2,133,977 nucleotides with an average GC content of 33.04%.
  • the genome comprises four replicons (Fig. 1), a circular chromosome of 1,827,111 nucleotides, a previously undescribed megaplasmid of 242,436 nucleotides designated pMPl 18, and two previously described plasmids of 20.4 kb QpSFl 18-20) and 44 kb (pSFl 18- 44) (21).
  • Multireplicon genome architecture may include minichromosomes, which are difficult to formally distinguish from megaplasmids [3].
  • pMPl 18 as a megaplasmid for the following reasons: It contains neither tRNA nor rRNA genes; it has plasmid-related replication and partition proteins; and it does not contain the only copy in the genome of any known essential gene.
  • the megaplasmid of the invention has a number of distinguishing properties. It is the first megaplasmid to be identified in a probiotic bacterium. It is the largest plasmid identified to date in gram-positive bacteria and it is the largest plasmid identified in the lactic acid bacteria (LAB) group to which L. salivarius belongs.
  • LAB lactic acid bacteria
  • pMPl 18 harbors an additional copy oirpsN (LSL_1944, ribosomal protein S14P), which is aparalog of the chromosomal gene LSL_1422 and is homologous to rpsN2 of L. plantarum and L.johnsonii.
  • the gene on pMPl 18 encoding a bile-salt hydrolase (choloylglycine hydrolase;
  • LSL_1801) is one of only two genes encoding bile-inactivating enzymes detected in the genome.
  • the copy number of pMPl 18 was estimated by PCR to be 4.7 _ 0.6 copies per chromosome equivalent in stationary phase, so gene dosage effects would contribute to amplifying the contribution of LSL l 801 to bile resistance.
  • the L. salivarius UCCl 18 chromosome harbors one copy each of ldhL and ldhD genes, whereas pMPl 18 encodes an additional copy of the ldhD gene (LSL_1887), whose product is 42% identical to the L. plantarum enzyme.
  • D-lactate is an important component of cell wall precursors in L.
  • LSL_1901 on pMPl 18 encodes a bifunctional acetaldehyde_alcohol dehydrogenase, which is the only enzyme in this strain that catalyzes the formation of ethanol from acetyl-CoA via acetaldehyde. Although not essential, the presence of this additional reductive pathway on pMPl 18 likely would improve the redox-balancing capability ofstrain UCC1 18.
  • L. salivarius comprises two subspecies, salivarius and salicinius [5], and both were found to contain megaplasmids (Fig. 8), of sizes varying from 100 kb to 380 kb. All of these megaplasmids hybridized with a pMPl 18 repA gene probe.
  • %A are chromosomal DNA, which caused nonspecific hybridization in Fig. SB in some samples). It is noteworthy that pMPl 18 contains a tract of genes that show low relatedness to known or suspected conjugation genes, thus representing a functional or remnant plasmid transfer locus. The ability to disseminate by conjugation would explain the apparently universal presence of pMPl 18-related plasmids (in strains so far tested), and this potential tra locus also might be involved in mobilization of smaller plasmids.
  • the megaplasmid pMPl 18 is the largest plasmid from lactic acid bacteria in current nucleotide databases. However, plasmids 100 kb in Lactobacillus spp. have been reported.
  • Lactacin F production by L. acidophilus strain 88 was shown by conjugation analysis to be linked to a 1 10-kb plasmid, pPM68 [6].
  • a plasmid of 150 kb was identified in L. gasseri by PFGE and was suggested to be linear on the basis of electrophoretic behavior [7]. However, its size and conformation were not confirmed.
  • salivarius UCCl 18 is the smallest Lactobacillus chromosome so far sequenced, 57.5 kb smaller than that of X. sakei and 165.5 kb smaller than that of L. johnsonii.
  • the existence of a circular chromosome is the default expectation for a conventional bacterial genome.
  • pMP 1 18 contains a repertoire of genes that likely confer metabolic flexibility, seen in the context of significant megaplasmid size variation in other strains, strongly suggests that the multireplicon genome architecture of Z. salivarius bestows on the species a dynamic and flexible genetic complement. This architecture could be in response to dietary fluctuations in host species, flexible niches in the GI tract, or adaptation to different hosts.
  • Lactobacillus salivarius strains were also examined for the presence of a megaplasmid (Fig. 3). All strains harboured a megaplasmid. The presence of the megaplasmid in a number of other strains of human origin with known probiotic properties (4) suggest that the megaplasmid contributes to the probiotic properties of Lactobacillus salivarius UCCl 18.
  • L. salivarius has an alanine dehydrogenase gene (EC 1.4.1.1; LSL_1768) located on the megaplasmid, which is unique among published Lactobacillus genomes but present in Lactobacillus casei and Lactobacillus delbrueckii draft genomes
  • L-alanine can be synthesized from pyruvate.
  • alanine dehydrogenase gene is a gene encoding a putative alanine permease (LSL_1767); both genes show elevated GC content (40.37% and 41.5% for LSL_1767 and LSL_1768, respectively), indicating lateral transfer.
  • the megaplasmid also encodes a paralog (LSL_1927) for one of the two enzymes required for conversion of pyruvate to L-aspartate.
  • the megaplasmid also harbors two genes (LSL_1931 and LSL_1932) predicted to encode the alpha and beta subunits of L-serine dehydratase (EC 4.3.1.17), which catalyzes pyruvateserine interconversion. Serine formed from pyruvate can subsequently be converted to glycine by a chromosomally encoded enzyme.
  • Serine may be thiolated to cysteine by CysK (EC 2.5.1.47; LSL_0026 and LSLJ 718), and cysteine can be converted to methionine by using four chromosomally encoded enzymes. Genes whose products are predicted to synthesize aspartate from pyruvate, and lysine and threonine from aspartate, were also annotated. Unlike L. plantarum, L. salivarius appears to lack the genes required for synthesis of tryptophan and related amino acids.
  • L. salivarius is currently regarded as homofermentative [5], meaning that sugars can be fermented only via the Embden-Meyerhof-Parnas pathway, and genes for the complete glycolysis pathway are present in the chromosome. Interestingly, genes for the pentose phosphate pathway also were found in the L. salivarius UCCl 18 genome, suggesting that it should be grouped among the facultatively
  • heterofermentative lactobacilli The genome sequence suggested that L. salivarius UCCl 18 would be able to assimilate ribose. This suggestion was experimentally confirmed by growth on ribose as a sole carbon source, and, furthermore, we detected lactate, acetate, and ethanol by HPLC in the culture medium, confirming the heterofermentative status of this strain.
  • LSL_1903 fructose biphosphatase
  • LSL_0395 chromosomally encoded phosphoenolpyruvate carboxykinase
  • pMPl 18 genes encoding enzymes involved in rhamnose and N-acetylneuraminic acid (sialic acid) catabolism as well as sorbitol utilization are present on pMPl 18.
  • pMPl 18 appears likely to increase the metabolic flexibility and, thus, competitiveness of L. salivarius UCCl 18.
  • the inferred properties of the Lactobacillus strain that are encoded by the megaplasmid include, but are not restricted to, the following: (locus numbers from genome sequence in parentheses):
  • LSLl 803 (SEQ ID No. 5) and linked genes). This would allow the bacterium to react quickly to changes in the gut environment, and thus confer a competitive advantage.
  • a plasmid transfer/conjugation operon (gene LSLl 812 (SEQ ID No. 6) and linked genes).
  • This cluster of genes may promote dissemination of the megaplasmid to other Lactobacilli, or may govern the mobilization of the smaller plasmids pSF-20 and pSF-44, both of which have genes which render them mobilizable, but are incapable of mobilizing themselves as they lack the transfer genes.
  • the megaplasmid could thus be a key element that may be exploited for introducing genes into other LAB, including by non-GMO methods such as protoplast fusion and conjugation.
  • LSLl 816 (SEQ ID No. 8), a putative fibrinogen-keratin binding protein.
  • LSL 1767 SEQ ID No. 9
  • linked dehydrogenase that may have a role in amino acid metabolism, or acid resistance, both of which are relevant for GI tract colonization and survival.
  • a bile salt hydrolase (coded LSL 1801 (SEQ ID No. 10)), which is expected to contribute to the resistance of Lactobacillus salivarius UCCl 18 to bile, conferring a survival and competitive advantage.
  • the predicted secreted protein complement of L. salivarius UCCl 18 contains 1 19 proteins. Among these 1 19 proteins, only 10 are likely sortase-dependent proteins.
  • the megaplasmid is predicted to encode eight of the secreted proteins. There is a single sortase gene srtA (LSL_1606) in the genome and genes encoding putative signal peptidase I (LSL_0876) and signal peptidase Il (LSL_0825).
  • Lactobacillus salivarius UCCl 18 the gene is srtA (LSL 1606).
  • Other Lactobacilli whose genomes have been sequenced and published (L. acidophilus, L. plant arum, L.johnsonii) also have only one sortase gene [[1 1 -13]].
  • sortase-dependant proteins The cell surface proteins that require the sortase enzyme for anchoring on the cell surface are referred to here as sortase-dependant proteins or SDP's.
  • Sortase acts upon the SDP's by recognizing sequence patterns at the carboxy-terminal end of the SDP, which it then bonds to the peptidoglycan in the cell wall [14]. These sequence patterns can be identified in genome sequences by for example computer analysis [15].
  • Table 2 The relationship of the SDP's of Lactobacillus salivarius UCCl 18 to other proteins is shown in Table 2.
  • AA amino acid
  • h for pseudogenes, the number of amino acids are indicated upstream and downstream of the internal stop codon, respectively; c bold font indicates a gene fragment; ''na, not applicable; "Blast hits were generated by comparing the 6-frame translation output of the target nucleotide sequence to a protein database using the BLAST-X algorithm; the protein accession number is indicated in parentheses, followed by the corresponding organism
  • LspB (LSLJ 085) SEQ ID No. 17
  • Adhesion is an important property of probiotic bacteria, as it allows them to interact with the host, and is thus implicated with colonization, immunomodulation and pathogen exclusion [16].
  • Cell surface proteins are the primary adhesion interface between bacteria and their host [17].
  • probiotic bacteria such as Lactobacillus salivarius UCCl 18
  • impairment of SDP anchoring would result in reduced adhesion to epithelial cells.
  • KO sortase gene knockout
  • Lactobacillus sakei is widely used for meat fermentation and food preservation, and thus is commonly used as a negative control for epithelial cell adhesion, as it is not a human GI tract-associated species [18].
  • the sortase gene KO strain though significantly less adherent than the parental Lactobacillus salivarius UCCl 18 strain, still adhered at around 50% of the wild-type level, and at almost double the level shown by L. sakei (Fig. 4). This indicated that proteins other than SDPs contributed to the intestinal cell adherence properties of Lactobacillus salivarius UCCl 18.
  • Sortase Independent Adhesins These putative sortase-independent adhesion proteins are referred to as Sortase Independent Adhesins or SIA's.
  • Bioinformatic approaches were used including searching for cell export signals (SIGNAL-P), Transmembrane Helix Markov Models (TMHMM) and homology scores against known or suspected adhesins in other bacteria to compile a list of SIA's.
  • Table 3 lists putative sortase-independent adhesins of Lactobacillus salivarius UCCl 18.
  • adhesion activity conferred by proteins identified in the Lactobacillus salivarius UCC 1 18 genome include the following.
  • the adhesin protein or derivative, fragment, or recombinant products thereof may be used for improving gut barrier function and or competitively excluding potential pathogens from binding to and or invading epithelial cells. They may also be used for mediating adherence of microorganisms to epithelial cells.
  • the protein may be used for the preparation of a medicament for use in generating an immune response, for engineering hyperadhesive mutants or for the preparation of a medicament for use in regulating cell cycle and/or invasive behaviour of tumour cells.
  • a deposit of a biologically pure culture of Lactobacillus salivarius strain UCCl 18 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) on November 27, 1996 and accorded the accession number NCIMB 40829.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • the strain of Lactobacillus salivarius is described in detail in WO98/35014.
  • the initial DNA sequence information was assembled into 110 scaffolds, organized into 18 contigs. To orient and link these contigs and to complete the genome sequence, approximately 500 inverse PCR reactions and 70 direct PCR reactions were performed. An additional 540 sequencing reactions were completed. Sequence data was assembled using Staden software, with in-built Phred and Phrap modules. Genes were predicted by a combination of standard openly-available software packages comprizing Orpheus, Glimmer, ZCurve 1.0, and Critica. These programs were run locally on a Linux-based server. Sequence analysis of the megaplasmid also showed that it had an asymmetric GC skew pattern (Fig. 1), which is typical of megaplasmids, especially those that are conjugative (ref [19]).
  • Example 2 DNA fragment analysis by Pulsed Field GeI Electrophoresis and Southern Hybridization.
  • Lactobacillus salivarius UCCl 18 to Pulsed-Field Gel Electrophoresis (PFGE), in combination with Southern hybridization.
  • PFGE Pulsed-Field Gel Electrophoresis
  • Sarkosyl N-Lauroylsacrosine
  • lysozyme proteinase
  • mutanolysin and
  • Aspergillus of ⁇ zae S 1 nuclease was purchased from Roche. Agarose gel plugs of high molecular weight DNA for PFGE were prepared by a modification of a published protocol. Bacteria were grown in MRS broth until it reached stationary phase. 1.5 ml cells was centrifuged (10,000 g, 1 min), washed once with 1 ml NT buffer (1 M NaCl, 10 mM Tris-HCl, pH 7.6) and re-pelleted. The cell pellet was resuspended in 450 ⁇ l NET buffer (1 M NaCl, 100 mM EDTA, 10 mM Tris-Cl, pH 7.6).
  • LMP low melting point
  • gel plugs Prior to incubation with selected restriction enzymes, gel plugs were cut into 3-mm slices with a sterile glass coverslip and soaked three times for 15 min in 1 ml 10 mM Tris-HCl, 0.1 mM EDTA (pH 8.0) at room temperature. Each slice was pre- incubated with 100 ⁇ l of restriction buffer recommended for the enzyme (New England Biolabs) for 30 min at room temperature and then replaced with 100 ⁇ l of fresh buffer containing 20 units of restriction enzyme. Restriction digests were carried out overnight at temperatures recommended by the supplier.
  • Plug slices were loaded directly into the wells of a 1% (w/v) pulsed-field grade agarose gel melted in 0.5 x TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.3) running buffer. The wells were sealed by melted 1% LMP agarose. DNA fragments were resolved using a CHEF-DR II pulsed-field system (Bio-Rad Laboratories) at 13 V/cm for 24 hours with 0.5 x TBE running buffer maintained at 1O 0 C. Linear ramped pulsed times were selected depending on the size of DNA fragments to be resolved. A frequently used protocol is 3 seconds to 80 seconds. Gels were stained in distilled water containing 0.5 ⁇ g/ml ethidium bromide for 60 min under dark conditions.
  • probe DNA was labeled with the enzyme horseradish peroxidase according to the instruction of the ECL direct nucleic acid labeling and detection kit (Amersham Biosciences).
  • ECL direct nucleic acid labeling and detection kit Amersham Biosciences.
  • Membranes were pre-hybridized in 10 ml ECL hybridization buffer containing 5% blocking agent and 0.3 M NaCl at 42 0 C for 30 min, followed which the labeled probe was added to the pre-hybridization buffer. Hybridization was done at 42 0 C for 16 h.
  • the megaplasmids of strains 43310 [NCIMB 41093] and 43324 [NCIMB 41044] were judged to be the same size as the megaplasmid of the invention, according to migration in PFGE.
  • Example 4 Gene knock-out construction by a two-plasmid integration strategy.
  • the regions flanking the sortase gene srtA (SEQ ID No. 30) of Lactobacillus salivarius UCCl 18 were amplified by PCR, ligated, and cloned into the pORI19 vector [20].
  • the resulting plasmid therefore contained a piece of DNA homologous to both sides of the srtAgene, but lacked the srtAgene itself.
  • the plasmid was introduced in Lactobacillus salivarius UCCl 18 cells harboring the plasmid pVE6007 which has a temperature sensitive origin of replication [20]. Integrants were selected by culturing the transformants in MRS broth plus erythromycin at 44°C, a restrictive temperature for pVE6007 replication.
  • the sortase KO thus lacks the sortase gene, but is otherwise identical to the parental Lactobacillus salivarius UCCl 18 strain.
  • Example 5 Assessment of adhesion of Lactobacillus salivarius UCC118 strains and derivatives to epithelial cells in vitro.
  • HT-29 enterocytic cell lines [21] were cultures as monolayers in DMEM (Dulbeccos modified Eagle's medium; Invitrogen) supplemented with 10% (w/v) foetal calf serum (Invitrogen). Cells were grown in 75 cm 2 tissue culture flasks (Costar, Cambridge, MA, USA) at 37°C in a humidified atmosphere containing 5% CO 2 . At 95% confluency the monolayers were passaged by incubating with 0.25% trypsin " (Invitrogen) for 10 min at 37°C. The adhesion of the strains was examined using a modified version of a previously described method.
  • HT29 cells were seeded at 1 x 10 6 cells / well in a 6 well plate (Greiner) and grown 10-12 days until confluence.
  • epithelial cells Prior to the assay, epithelial cells were washed once in serum-free DMEM. Bacteria were washed once in sterile PBS and suspended to 1 x 10 8 CFU/ml in PBS. ImI bacterial suspension was combined with ImI serum-free DMEM and added to the epithelial cells (MOI - 50:1) and bacteria were allowed to adhere for 30 min at 37°C in a humidified atmosphere containing 5% CO 2 . Unadhered bacteria were removed by washing the cells five times using sterile PBS.
  • Epithelial cells with adhering bacteria were scraped off using a rubber scraper (Greiner) and resuspended in ImI PBS. Serial dilutions were plated on MRS agar (Oxoid) and percentage adhesion was determined. Adhesion of mutants was expressed relative to that of wild type where wild type was set to 100%.
  • Example 6 Identification of cell wall-anchored proteins.
  • L. salivarius UCCl 18 possesses the second-largest genome (2.13 Mb) of the fully sequenced lactobacilli and is the only sequenced Lactobacillus strain harboring a megaplasmid [22].
  • a megaplasmid [22].
  • pMPl 18 the megaplasmid
  • two are encoded by the 44-kb plasmid pSFl 18-44
  • one is encoded by the 20-kb plasmid pSFl 18-20.
  • Deduced products of an additional five pseudogenes were predicted to be secreted, with two encoded by the chromosome and three encoded by pMPl 18.
  • sortase substrates for L. plantarum and L. johnsonii have been described previously [23].
  • LSL_0152 encodes a protein which shares 30% identity with the mucus-binding protein Mub of L. reuteri.
  • LSL Ol 52 is interrupted by a stop codon (TGA) at nucleotide position 499.
  • TGA stop codon
  • LSL_1319 shows 21% identity to the R28 protein of Streptococcus pyogenes , which is involved in binding to epithelial cells.
  • the DNA sequence of LSL_1319 is interrupted by a stop codon (TAA) at nucleotide position 667.
  • TAA stop codon
  • the third pseudogene, LSL_2020b is located on pSFl 18-44.
  • LSL_2020b is interrupted after 1,938 base pairs by the stop codon TAA.
  • TAA stop codon
  • pseudogenes there is no evidence of a second ribosome binding site with a start codon, which could lead to translation of the distal fragment of the gene.
  • the other pseudogenes are disrupted by a stop codon, we identified a pseudogene (LSL_1774b) with a frameshift in its sequence which introduced a stop codon.
  • LSL_1774b The product of LSL_1774b is homologous (32% identity) to a 1 ,480-amino-acid proteinase (PrtR) of the human isolate L. rhamnosus BGTlO. Apart from the pseudogenes, two gene fragments harboring a sortase recognition sequence were also identified (Table 5). Both gene fragments are located on pMPl 18.
  • LSL_1832b is a 2.3-kb gene fragment whose derived amino acid sequence harbors an LPQMG sortase recognition motif. The fragment is homologous (17% identity) to the C- terminal region of a 1 ,575-amino-acid salivary agglutinin-binding protein of Streptococcus gordonii. The smallest gene fragment harboring a sortase recognition motif is LSL_1902b (147 base pairs). It has no homology with proteins in the nonredundant BLAST database.
  • LspA (LSL_0311) is a 1 ,209-amino-acid protein which contains seven repeats of 79 amino acids (Rl to R7) (Fig. 9). Rl and R7 are the least conserved repeats, sharing 73% identity, whereas R2 to R6 are more conserved, sharing 92% identity. Pfam analysis revealed that each of these repeats is similar to mucus-binding domains
  • LspB is an 827-amino-acid protein (Fig. 9) containing an LPQMG cleavage motif. Three 13-amino-acid repeats were identified at the C-terminal end of the protein. The repeats are 100% identical, and Pfam analysis revealed no predicted function.
  • LspB enterococcal surface protein (Esp) of Enterococcus faecium (AAQ89938) which has no assigned function (Fig. 9).
  • LspC LSLJ 335) is 785 amino acids in size and has four repeats of 97 amino acids (Fig. 9). There is over 98% identity among these repeats, and their sequences are similar to those of mucus- binding domains, as predicted by Pfam analysis, with E values ranging between 10 3 and 10_4 but with all scores being above the gathering threshold. It is homologous to the 3,269- amino-acid mucus-binding protein (Mub [AAF25576]) previously characterized in L. reuteri (42).
  • sortase-dependent proteins play a role in adhesion and virulence in a range of organisms .
  • a sortase-dependent protein(s) in L. salivarius UCCl 18 is involved in adhesion, we constructed a mutant strain lacking the
  • sortase gene (LSL_1606).
  • the small size of sortase did not allow us to disrupt the gene by plasmid integration, and we therefore opted for a gene deletion, using a double-crossover strategy.
  • Upstream and downstream flanking regions of 772 bp and 818 bp, respectively, were amplified.
  • the upstream flanking amplicon includes the first 13 codons of the sortase gene, whereas the downstream flanking amplicon includes the last 3 codons.
  • Both flanking amplicons were joined by SOE-PCR and cloned into pORI19.
  • the resultant recombinant plasmid, pLSOOl was transformed into L. salivarius UCC 118 harboring p VE6007, and a double-crossover mutant was obtained.
  • the deletion of the sortase gene in strain UCCl 18 was verified by
  • Example 9 The sortase mutant has reduced adhesion to epithelial cells.
  • UCCl l&jsrtA Following the construction of UCCl l&jsrtA, we tested the strain for adhesion to intestinal epithelial cells. UCCl l8_srtA adhered significantly less to HT29 cells (P _ 0.04) than the wild-type strain did (Fig. 5A). We also employed a semiquantitative real-time PCR method to validate the viable count method (Fig. 5B). The adhesion of the UCC 11 S_srtA mutant was also significantly reduced (P _ 0.04) as measured by this method, at 61% of the level of the wild-type strain.
  • Example 10 Transcriptional analysis of sortase-dependent proteins.
  • RT-PCR endpoint reverse transcription-PCR
  • Example 11 Role in adherence of LspA, LspB, and LspD.
  • the chromosome of L. salivarius UCCl 18 potentially encodes 108 secreted proteins, which comprise 6.2% of the chromosomally located open reading frames (ORFs). This is lower than the proportions for L. acidophilus, L. plantarum, L. sakei, and L. johnsonii, all of which devote 7% of their coding capacities to secreted proteins.
  • ORFs open reading frames
  • the two secreted proteins encoded by pMPl 18 with assigned functions are an oligopeptide binding protein and an amino acid transporter.
  • the plasmids pSFl 18-20 and pSFl 18-44 contribute little to the predicted L. salivarius UCCl 18 secretome, encoding one and two secreted proteins, respectively.
  • Ten proteins were identified in L. salivarius UCCl 18 as sortase substrates by manual screening, and searching the genome with a hidden Markov model (40) did not identify additional potential SDPs.
  • two sortase substrates were encoded by gene fragments and four were encoded by pseudogenes interrupted by a single stop codon or frameshift.
  • the megaplasmid may be used to transform other probiotic lactobacilli and thus improve or manipulate their desirable properties, including but not restricted to growth rate in synthetic medium (this refers to the sugar utilization genes on the megaplasmid), gut colonization, gut persistence, host immunomodulation.
  • the pMPl 18 or other lactobacillus megaplasmids may be used to mobilize other plasmids into recipent strains including, but not restricted to, other lactic acid bacteria.
  • Derivatives may be made of the replication origin of pMPl 18, with and without the putative conjugation-related genes, and these derivative plasmids may be used as cloning vectors in Lactobacillus sp.. or other Gram-positive bacteria.
  • UCCl 18 or other lactic acid bacteria may be manipulated based upon knowledge of the surface proteins as hereinbefore described.
  • the gut colonization, gut persistence, host immuno-modulation of UCCl 18 or other lactic acid bacteria may be modulated based upon knowledge of surface proteins as hereinbefore described.
  • genes and corresponding proteins as hereinbefore described may be used to confer de novo gut interaction properties, or to improve the gut interaction of heterologous bacterial species
  • Genes from pMPl 18 and the UCCl 18 chromosome may be used or manipulated to modulate clumping of the cells of UCCl 18 or other lactic acid bacteria.
  • Genes from pMPl 18 and the UCCl 18 chromosome may be used or manipulated to modulate or alter the metabolism of UCCl 18 or other lactic acid bacteria.
  • Lam M. Pallen, and TJ. Foster, Characterization of novel LPXTG- containing proteins of Staphylococcus aureus identified fi'om genome sequences. Microbiology, 2003. 149(Pt 3): p. 643-54.
  • differentiated HT-29 cells a basis for host-pathogen interactions in the gut. Infect. Immun., 1989. 57(12): p. 3727-34.
  • SEQ ID No.2 The circular chromosome of 1 ,827, 111 bp. (CP000233)

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Abstract

La présente invention concerne un polynucléotide isolé comprenant une séquence d'acides nucléiques, représentée par le numéro d'identification SEQ ID No. 1, ou un variant ou un fragment de cette séquence. L'invention concerne également un polynucléotide isolé comprenant une séquence d'acides nucléiques, représentée par les numéros d'identification SEQ ID No 2 à SEQ ID No. 30, ou un variant ou un fragment de cette séquence.
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* Cited by examiner, † Cited by third party
Title
CLAESSON M J ET AL: "Multireplicon genome architecture of Lactobacillus salivarius" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 103, no. 17, April 2006 (2006-04), pages 6718-6723, XP002402646 ISSN: 0027-8424 *
FLYNN SARAH ET AL: "Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118" MICROBIOLOGY (READING), vol. 148, no. 4, April 2002 (2002-04), pages 973-984, XP002410387 ISSN: 1350-0872 *
UNIVERSITY COLLEGE CORK, MICROBIOLOGY, IRELAND, CORK: "Lactobacillus salivarius subsp. salivarius UCC118 plasmid pSF118-20, complete sequence"[Online] 17 December 2004 (2004-12-17), XP002410388 Retrieved from the Internet: URL:http://www.ncbi.nlm.nih.gov/entrez/que ry.fcgi?db=genome&cmd=Retrieve&dopt=Overvi ew&list_uids=18106> cited in the application *
UNIVERSITY COLLEGE CORK, MICROBIOLOGY, IRELAND, CORK: "Lactobacillus salivarius subsp. salivarius UCC118 plasmid pSF118-44, complete sequence"[Online] 17 December 2004 (2004-12-17), XP002410389 Retrieved from the Internet: URL:http://www.ncbi.nlm.nih.gov/entrez/que ry.fcgi?db=genome&cmd=Retrieve&dopt=Overvi ew&list_uids=18107> cited in the application *

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Publication number Priority date Publication date Assignee Title
WO2019169181A1 (fr) * 2018-02-28 2019-09-06 Evelo Biosciences, Inc. Compositions et méthodes de traitement du cancer à l'aide de lactobacillus salivarius

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