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Title:
LISTERIA MONOCYTOGENES CYTOTOXIN LISTERIOLYSIN S
Document Type and Number:
WIPO Patent Application WO/2009/092790
Kind Code:
A1
Abstract:
The invention relates to a gene derivable from the LIPI-3 pathogenicity island of the gram positive pathogen Listeria monocytogenes and to the Listeria monocytogenes cytotoxin, Listeriolysin S. The present invention further relates to the application of Listeriolysin S as a target for diagnostic screening and as a therapeutic agent such as an anti-cancer agent.

Inventors:
ROSS, Paul (Moorepark Research Centre Fermoy, Cork, IE)
HILL, Colin (CorkCollege Road, Cork, IE)
COTTER, Paul (CorkCollege Road, Cork, IE)
Application Number:
EP2009/050774
Publication Date:
July 30, 2009
Filing Date:
January 23, 2009
Export Citation:
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Assignee:
TEAGASC, THE AGRICULTURE AND FOOD DEVELOPMENT AUTHORITY (19 Sandymount Avenue, Dublin, 4, IE)
UNIVERSITY COLLEGE CORK - NATIONAL UNIVERSITY OF IRELAND, CORK (College Road, Cork, IE)
ROSS, Paul (Moorepark Research Centre Fermoy, Cork, IE)
HILL, Colin (CorkCollege Road, Cork, IE)
COTTER, Paul (CorkCollege Road, Cork, IE)
International Classes:
C07K14/195; A61K38/16; C12N15/00; C12Q1/68
Attorney, Agent or Firm:
GATES, Marie Christina Esther (Tomkins & Co, 5 Dartmouth RoadDublin, 6, IE)
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Claims:

Claims

1. A gene derivable from the LIP 1-3 pathogenicity island of the gram positive pathogen Listeria monocytogenes , the island being flanked by two Rho-independent terminators, located after Imof2365_l 111 and HsP, respectively, and two Rho- independent duplicated glyoxalase-encoding genes (Imof2365_llll, 1121).

2. A gene as claimed in claim 1 wherein the island is downstream of lmofi 365 _1110 and upstream of Imof2365_l 127. are located up- and down-stream of the island respectively.

3. A gene as claimed in claim 1 selected from the genes HsA, HsG, HsH, HsX, HsB, HsY, HsD and llsP.

4. A gene as claimed in any of claims 1 to 3 selected from SEQ ID NOs. 3, 4, 5 7, 9, 1 1, 13,15, 17and 19 or sequences substantially similar thereto.

5. A gene as claimed in any preceding claim which is HsA.

6. A gene as claimed in any preceding claim wherein the sequence is associated with a subset of lineage I strains of Listeria monocytogenes.

7. A nucleic acid sequence as set forth in SEQ ID No. 1 or a sequence substantially similar thereto which also encodes a cytotoxin

8. An amino acid sequence of SEQ ID No. 2 or a sequence substantially similar thereto which also encodes cytotoxic activity.

9. A cytotoxin, Listeriolysin S, wherein the cytotoxin is haemolytic to sheep red blood cells, cytotoxic to C2-Bbe 1 , J774 and CT-26 cells and contributes to survival of the pathogen following polymhorphonuclear neutrophils (PMN) challenge and to virulence, as assessed by murine assays.

10. A cytotoxin as claimed in claim 9 wherein the toxin confers a high virulence potential.

1 1. A cytotoxin as claimed in claim 9 or 10, for use in the preparation of a medicament for the treatment of disease.

12. An amino-acid sequence selected from the group consisting of SEQ ID NO 6, 8, 10, 12, 14, 16,18, and 20.

13. A diagnostic kit for distinguishing between high and low virulent strains of Listeria monocytogenes comprising a gene as claimed in any of claims 1 to 6 or a sequence of claims 7, 8 or 12.

14. A kit as claimed in claim 13, further comprising probes based on the genes of claims 1 to 7, and / or antibodies raised against a the product of a gene as claimed in any of claims 1 to 7 or an amino-acid sequence of claims 8 or 12.

15. A pharmaceutical composition comprising the cytotoxin of claims 9 to 11, the gene of claims 1 to 6 or a sequence of claims 7, 8 or 12.

16. A disinfectant composition comprising the cytotoxin of claims 9 to 11 , the gene of claims 1 to 6 or a sequence of claims 7, 8 or 12.

17. A gene substantially as described herein with reference to the Examples.

18. An antibody raised against a the product of a gene as claimed in any of claims 1 to 7 or an amino-acid sequence of claims 8 or 12

19. A nucleic acid sequence substantially as described herein with reference to the Examples.

20. An amino acid sequence substantially as described herein with reference to the Examples.

21. A cytotoxin substantially as described herein with reference to the Examples.

22. A diagnostic kit substantially as described herein with reference to the Examples. 22. A composition substantially as described herein with reference to the Examples.

Description:

Title

Listeria monocytogenes cytotoxin Listeriolysin S Field of the Invention

The current invention relates to bacterial cytotoxins. More specifically the present invention relates to a Listeria monocytogenes cytotoxin, Listeriolysin S. The present invention further relates to the application of Listeriolysin S as a target for diagnostic screening and as a therapeutic agent such as an anti-cancer agent. Background to the Invention

Listeria monocytogenes is a food-borne gram-positive pathogenic bacterium responsible for listeriosis, a life -threatening infection of pregnant and immuno -compromised humans. Strains of Z. monocytogenes can be separated into three evolutionary lineages (I-III), all of which are capable of causing disease in humans and animals.

Lineage I-lineage in all possess the well-established virulence genes associated with the pathogenesis and intracellular life cycle of Z. monocytogenes. These genes are located on a 9-kb virulence gene island designated Listeria monocytogenes Pathogenicity Island 1 (LIPI-I). LIPI-I contains six genes, all of which are directly involved in pathogenesis. The gene My located on LIPI-I encodes Listeriolysin O (LLO), a pore forming thiol- activated toxin secreted by Z. monocytogenes . The intracellular life cycle of Z. monocytogenes begins with adhesion and subsequent penetration into a host eurkaryotic cell. At this point during the initial entry into the cell the bacterium becomes engulfed by a host vacuole. The disruption and escape from the host vacuole by the bacterium is essential for intracellular survival and proliferation. It is at this point that LLO is considered to play an essential role, facilitating the survival of the bacterium in an infected cell. All L. monocytogenes strains, regardless of lineage, produce Listeriolysin O. It is well known in the art that lineage I strains (consisting primarily of strains of serotype l/2b and 4b) are responsible for the vast majority of outbreaks of listeriosis and almost two thirds of human sporadic cases. This lineage includes three previously defined epidemic clones responsible for multiple listeriosis outbreaks in Europe and North America.

However, the genetic basis for the enhanced virulence of lineage I strains has not been established. It is not possible to reliably discriminate between "epidemic" and "non-

epidemic" strains of the disease in animal models. Furthermore, on the basis that there have been a few exceptional outbreaks by non-lineage I strains and that these stains are also responsible for the remaining third of sporadic cases, it is evident that should a lineage I-specific factor exist its presence is not a pre -requisite for pathogenesis but rather an indication of a higher virulence potential.

A previous study by Nelson et al revealed there to be relatively little genetic difference between the high and low virulence strains of Z. monocytogenes . For example, there are a mere 51 genes present in serotype 4b strains (F2365, H7858; lineage I), which are lacking from their l/2a (EGDe, F6854; lineage II) counterparts. To date, however, no candidate genes have been proposed which could explain the enhanced virulence of lineage I L. monocytogenes strains.

A variety of laboratory procedures have been developed in an attempt to accurately determine the subtype of Z. monocytogenes strains. These methods have been principally serological (e.g. Listeria serotyping antiserum SEIKEN; Seiken Co. Ltd., Tokyo, Japan) and nucleic acid based techniques including PCR (e.g. multiplex PCR can discriminate between strains from different lineages (I)) DNA sequencing (e.g. multi-locus sequence typing (MLST) has been used to differentiate between and within lineages (2-4), hybridization (e.g. single nucleotide polymorphism (SNP)-typing has been used to identify different sequence types and multilocus genotypes (MLGTs) within specific lineages (5)), pulse field gel electrophoresis (e.g. to differentiate between different patterns among 4b strains (6)) and ribotyping (e.g. subtyping within lineages (7, 8)). Although these methods have proven successful at differentiating between the or within different lineages of Z. monocytogenes, they are not effective at determining the virulence potential of Z. monocytogenes strains as highly virulent strains can be found within each lineage.

The identification and differentiation of Z. monocytogenes strains is a key problem facing the food and medical industries. Therefore, the availability of a diagnostic kit with the potential to differentiate between high and low virulence strains would be of great benefit to food producers, vets and clinicians. This in tandem will allow legislation to adjust the levels of Z. monocytogeneous that are permitted in food in order to effectively control levels of Z. monocytogenes and hence to prevent infection of listeriosis.

The Object of the Invention

It is an object of the current invention to provide a cytotoxic agent, which can be used as a therapeutic agent. Another object is to provide a lineage I specific cytolytic virulence factor, called listeriolysin (LIs) S and its use as a target for diagnostic screening. LIs detection can be used to distinguish between high and low virulence L. monocytogenes strains. It is a further object of the invention to provide a therapeutic agent for the treatment of disease. LIs or bacteria producing LIs are cytotoxic to a number of cell types including cancerous cells. LIs has an application as a chemo therapeutic agent, potentially acting as an alternative to existing agents e.g. bleomycin (treatment of Hodgkin lymphoma, squamous cell carcinomas, and testicular cancer, pleuodesis and plantar warts) and epothilone (a novel anti-cancer peptide currently undergoing clinical trial).

Summary of the Invention

According to the present invention there is provided a gene derivable from the LIPI-3 pathogenicity island of the gram positive pathogen Listeria monocytogenes, the island being flanked by two Rho-independent terminators, located after Imof2365_l 1 11 and HsP, respectively, and two Rho-independent duplicated glyoxalase-encoding genes (ImoJ2365_llll, 1121). The island is downstream of lmofi 365 _1110 and upstream of Imof2365_l 127. The location of these genes is significant as corresponding genes have been identified at a similar location within the genomes of all L. monocytogenes sequenced to date. The gene may be selected from the genes HsA, HsG, HsH, HsX, HsB, HsY, HsD and HsP. The gene may be associated with a subset of lineage I strains of Listeria monocytogenes.

The current invention also provides a nucleic acid sequence having the sequence of SEQ ID No. 1 or a fragment of SEQ ID No. 1 , or a sequence substantially similar thereto also encoding a cytotoxic agent. The invention also provides an amino acid sequence corresponding to the above nucleic acid sequence having SEQ ID No. 2 or a sequence substantially similar thereto also encoding a cytotoxic agent. SEQ ID No. 1:

ATGAATATTAAATCACAATCATCAAATGGCTACAGTAATAATGCTGTAGGCT CTGAAGCAATGAACTATGCAGCTGGATGTTGCTCATGTTCTTGTTCAACTTGC ACATGTACATGTACATGCGCATCATCTGCTGCAACCAAAATGTAA

SEQ ID No.2:

MNIKSQSSNGYSNNAVGSEAMNYAAGCCSCSTCTCTCTCASSAATKM.

The sequence encodes a cytotoxin. The nucleic acid sequence is located on a pathogenicity island of the gram-positive pathogen Listeria monocytogenes referred to as LIPI-3. LIPI-3 contains the 8 genes UsA, HsG, HsH, UsX, UsB, UsY, UsD and UsP. The 8- gene region is flanked by two Rho-independent terminators, located after Imof2365_l 11 1 and HsP, respectively, and two Rho-independent duplicated glyoxalase-encoding genes (ImoJ2365_l 111, _1121). The outer limits of the LIPI-3 are also apparent from comparison with the genome sequences of strains that lack the island i.e. a number of these strains possess genes corresponding Imof2365_1111 and _1121 but none possess any of the Hs genes. LIPI-3 is also distinguished by having a very high genomic dissimilarity value (δ* of 0.1178) relative to that of the remainder of the F2365 genome (δ* of 0.0343) and an atypical %GC content (29.9 vs 38%).

The invention also provides a nucleic acid sequence selected from SEQ ID NO. 3, 4, 5 7, 9, 11, 13,15, 17and 19 and an amino-acid sequence selected from SEQ ID NO 6, 8, 10, 12, 14, 16,18, and 20 and sequences which are substantially similar thereto. By 'substantially similar thereto' is meant sequences which are homologous to the specified sequences, particularly sequences which are about 70% homologous under low stringency conditions, or about 80% homologous under medium stringency conditions, or about 90% or about 95% homologous thereto under high stringency conditions. 'Stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that "stringency" conditions may be altered by varying the parameters just described either individually or in concert. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (for example, hybridization under "high stringency" conditions, may occur between homologs with about 85-100% identity, preferably about 70-100% identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (for example, hybridization under "medium stringency" conditions may occur between homologs with

about 50-70% identity). Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. "High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5xSSPE (43.8g/l NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and lOOμg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0. IxSSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length is employed. "Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5Xsspe (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.OxSSPE, 1.0% SDS at 42° C, when a probe of about 500 nucleotides in length is employed. 'Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5xSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5xDenhardt's reagent [50xDenhardt's contains per 500ml: 5g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5xSSPE, 0.1% SDS at 42° C, when a probe of about 500 nucleotides in length is employed.

SEQ ID NO 3; LIPI-3

GTGGAGTGAAATATAAGTTAGAGATTATTTTTCGATTAGGTGTACTTTTTTTTT

GTCTTCGTTTTATAGATTTTAGTATTTTTTTAATCTTGTATTATTAATGATAGTT

ATCTATAGCTTATATGCAGATTATGGAGTTGTATTAGTAAAAAAGCCCTGAA

GCCTCAGCATTATTGAGTTTCTAGGGCTTTTTTATGTCGAGTGATAACCGTTTT

TTATTATCGTACTATTTTTGTGGATAATATTTTGTCCAATTATACATATATACT

TTTGGAGATATTTAAACCAATTTTGCATATTATCAAACGGAGGGATATATTTT

TTATAAAAATAATTAAAAAAATTATTTAATTTTCTGAAATAAACAAAGAATTT

ATTTATTTTGATAATTATATTGAAAACGATTTCACAATGTGATAGGATGAACT

AAGGGATAATTTATTCCAAAAAATAAAAAGGAGGCATTTGAATGAATATTAA

ATCACAATCATCAAATGGCTACAGTAATAATGCTGTAGGCTCTGAAGCAATG

AACTATGCAGCTGGATGTTGCTCATGTTCTTGTTCAACTTGCACATGTACATG

TACATGCGCATCATCTGCTGCAACCAAAATGTAAATTTTAGAATGAAAAAGG

GATACATTTGTGTCCCTTTTCAATAAGTTATGAGGTGCATGTGCATATGAATA

ATATTTTGGAAACGAAAAATTTGAAAGTTACAATAAATAATAAAGTAATTCT

ATATTTAGATAAAGAAGTTTGTATTTCAGAAAAAGACAAAGTTGCCATTCTA

GGAGACAATGGAGCTGGGAAAACCACGTTAGTAAATAGTATTCTTGGTGAAA

AGAACTCTTCTGGAGAAATTACCAAAAAGTTTAAAAAAAATGACTGTGGTGT

AGTGTTTCAAGAAAATGCATATAATGATTTGATGAAAGTTTATGAATTGATTA

CTTTAGTTCTGCCACATCTAAAGAAAAAAGAGAGAGCGCAGTTTTTACACAA

ATATGAACTTGAAAGTTTGAGAAAAAAGTACATTAAAGACTTATCTGGTGGA

GAAAAACAACGACTAACACTATGTTTAGTACTAGAAAGTCATAAGAAATTAT

ATATCTTTGATGAGTTAACTTCAGGATTAGATTATAAGAAGAGACTAGGTTTA

CTTGCTTTGATGAAAGAAAAAACTAAGGACGCGACGGTGATAAATATAACGC

ATTACTTTGAGGAAGTTGAAAACTGGGCAACGAAAGTTTTGATTCTTCAAAA

AGGAATTTTACTTTTTTGGGGAACAATATCAGAATTTTTCTCTAATTTTCCTCA

TTATTCTGTTATTAAAGTAGATCAAATCGAACTAACAAAGATAGATGAAACA

GATATGACTTTTATGCAAAGCACAGATACAGGTGACGGGATAGCAGTTATTT

GTTCGGATTTGCAAATTCAGGAAGAGACAAAGAAAATTTTGGATAAAAAAAA

CGTCACATACAACACAATAAAACAAAATATATATACAACTTATTTAGTTGCA

TATTTGCGCGGAACATCAAGCAGTGAACAGGAGGTACTGATATAATGAGTTA

TTTATGGACAAGTATTAAAATGCAATTTAGAATTCCAGTATCTGTGTTTTTCT

CATTATTATTTCCACTAATCATGATGTTCGCTATGGTTACTTCTTATGGAAACT

TTGATATTGGTGAAGGTTATCATTTTGTAGATAAATATTTTTTGATTAGTACA

GGGATGGGCATGTTACCAATAGCATTAATCAGTTTTCCAATCTGGCTTGGTGA

AAGTGTTCAAAATAAAAGCTATAAAAGATTAGAATATTTTGGATTGAGTGCA

CAGAAAATAATTGTTTCAGATGTATGTTCTTATATTTTGCTAACAGCCTTAAG

TATTTTTGTCAATATACTGTTTGGCTACTTGGTATATGGACTACATATCCCAG

ACTGGCAATATTTTATTGCTTATGTTTTGCAATGTCTGTATTGTAATCTAGTGC

TTTTGATTTTTGGTGCCTTGCTTGCTTTAATTATTAAAAATCCCCGGATATTGA

TGCCAGTAGGAATGTGTTTATTATTTATGTTTTATATTTTTACAGGAACATTTT

CATCTTTTTCGGAACTTCCTAAATCATTTCAAGCAGTTGGTAATTTTTTACCTA

TGAAATATATAATGAATGATTTATTTAATGTTTGGACACAAAATAAATTGTTC

ATCTCTAAGTTTTTAGCTTTAAACACGTTATATGGAATCATCTTATCATTCGCA

CTAGTTGCTTTTTTGTTGAGGCAAAAGAAAATAAAAATTAAACCATAATTTAT

TATACTCAAGGTATAGAAAGGATTGTTTGTAATCTATGAAAAAAAAATTTAG

TAATCCCACGTTTAGAATTATTGCATCAATTGTTCTAGGGATTTTGATAGGTG

TTCTTATTTGCTTTGTGGCTATTGGTTTAGGTTACATTCACATGAATGATGGCA

CATTAAAAGAGTATAGT GTGAAAATTTT CGG ACT AACT ATTTTTGAT ATT AAA

AGAGTT GGTAGTGAGATGGT GGGAACACCTAATAACACTAGCAT GATGTTTA

TAGGGGTTATTATTTCTATGATACTAGCTATTGTTGTAGAAATTATTGTTTCGT

TAAAAAACAGACATAGAAAGGAAACAGCAAAATGATCGACTATGAGAAAAA

AGGCTTTTTTAACATCCACACATTGGTAAATAAAGATAATGCTAATATTTCTA

ATAGTGATAATAAACATATTTATTCCCAGCTGATGTCTGGTAATGGCAATTCA

CCAATGCTAGGTTATTTATTAAATATGAATAAACAAAACTTGAATGACTTCAA

AAGTATCATGTTTTATAATGAATCTAATCTAGCTTCTTTAATTAACGAGGCAA

GAGAAATGGAAGAATTAATAGATAGCTCAACTCTTTTTTTAAGTAAAACAAA

TAAAAAAATTAAAACCCATTTTTCTAAAGTGCTTGAGCAAAGACATAGTACA

AGAAATTTTGTATACGAAACCATGGATTTATCAACATTCTCAAATATCATACA

GTTTTCTTTTGGCCTTAGTACAAGAAAGTTGGTTTATAACGACTTACAATCAA

CCACCAGGCATTATTCTTCTGGGGGCGGTTTATATCCAATTGATGTTTTTTTGT

ATATAAATAATATTTCTGGAATTGCCAAGGGTATATATAAATATCAGCCATAC

ACGCATAGCTTGCATCCTCTAGATGTAGATAAAATTGATGTAGAGTCGTTTTT

CGTAGGTGATAATATTGACACTTCTAATATGAATTTTTGTGTTTTCTTTGGATA

CTCTATTAATAAAAACTATGTGAAATATGGAGAACTATCTTTACTGAATACGT

TTGTGGAACTAGGAGGAATATCACACAACTTTGATTTAGTTTGTCATTCGGTT

CACTATACAAGTTGTCCTATTGCAGGTTTTAACAAGTCGTACATAGAGAAGCT

TTTATATTTAGATGGGATAAATGATCACATTATATTTACAAATATTTGTGGAA

AGGAATGATATTTTTATGAAAAATTTTGATATTAGAATAGGAACGCAGACAA

TTGACAATGATACGGAATTCATTTTAAAAAGAGGGGTTATTCATAAGAATGA

GATCGTTATCAATAAAGAGGAAAGTTCAAAAGAATTTGTTTCAACATTTAAA

GAATTGGTCAAGAAAAAAACTATTACTATATCTTCAGAAGATGCAATCTACA

ATGATTTTGAAACACTTACAAAATTTGGCTTTTTAACAATTTCTAAAAACCAA

ACCTTAAAACCTCTCTTGATAGTTGAAGATGCTTTGTTTGATGACGTGAAGAG

CTATTTCCAGGAAGAAATTGAGATTTTATCATCCTCTGAATTTCTTTTAAAAA

AAGATATTCGGTTACTTACTGAGAACAAAGATATATTGCAACTTACTAAACT

AGTAGATGAAAAAAAGGAATTAATGAAAAATTATAATTATATTTATTTAATC

ACAAACATTTCCAATATTTCCTTATTACGTGGATTTAACAAGTTAATGAAAGA

GACTAACAGTGTTAATACCATCGCATTTTTTGATAATGAAAATGTATTTGTTA

CTTGTATAGAACATGGAGAAACTGGCTGCTATGAGTGTTTAGAGCGGCAACT

ACTCTCTCATTTTGATGGTGTTGTTACGGATTATTTAGTACAGTCAGAAAATA

ATGTTTCCACAGCTGAATTGATGTTTGTTCTTTCAATCATAAAAAAAGAAATC

GAAAATACCTCTATTTATGGTCAATCTTCTCTGTTAGGCAATCTTCTCCACTTT

AATTTTAATAACTACGAGTATACGTTTAATACAAATAGAATCCAAAGTTGTTG

TTCTACCTGTGCTACTTTCAATAATATATTATTTGAAGAGCAAAACATTCGGT

CAGTGAATATACTAAAGGAGCTGATGAGTAGTGATTAGCATACAAAATAATT

TGGAATATAACAAGTTACGTTGGGAAACTTTGAGTGGAAATGTAACAGGAAT

ATGGGAGAATAATAAGTTCTTTCTAGGCTCTAGTTCTTACCCTATTATGAAGT

ATCATTACATTACAGCTAATTTTGTTAATTTTGAAAAACATATTTCTGAAAAT

ATGCCTAAAATAAGTTACCATTTAAGTGGATATGGTGTTAATTTTAATGAAGC

GCTTGTTAGTTTTATTGGTGAAAGCGCAGAGAGATATACATATTCCTTACTAC

CTACTATTATTAAGGACAGGATTATTTTTAGATCATATGAAGAAATGACAAA

AGAATATAAAACTGACTTAATATGTGAACTTAAATACATAAATTCGTATTACT

CTTCTGAAGTATGCGAAAATTATGTTACTCCAAATGATACTATTCAATGGATA

GCAATGAATTCTCTTGTTCATTCTGATAAAAAAGTATGGATGCCTTTGCAATT

TGTTACTATGTATACAGAAGAAATGTTTTCTAATGAAAAAAGATATGTAACT

AGCGCTGTATCAACAGGCACTGCTTGTCATGAAACTGTAGAAAAAAGTATAG

AAAATGCCCTAATTGAATATCTTCAAATTGATTCCTTTAATTTATGGTGGTAT

GGAGGGTTTCGTGCGAGAGATATAGAAATAGATATCACTCGAAATATATCAA

GCTGGTTCGACAATCAAGTAGCTGTGAAAAAATTTTTATCAAAGTTTAATGTA

CATTTTTCAGATATAAGTTTTGATAAATCAATTTATATTGTGCTATGCGAAAT

AGAGGCAAAAAATTCAAGTGATGCCTTTCCTAAATATACTGTTGGCGTTCAA

GGCGGATATTCACTAGATAAATCAATATATCGTGCATTTATGGAATGTCTAAC

TGTACTAGAATATAATATGAATGTCACTTGGACAGATAAAGAGAAATTTCTTT

CAGTCACACAGGAAACACGTGTTATAGATAATTTGGATGATAATGTTATTTAT

TATTCAAAGTATGGAAAACCAGAATTGCAATATAATACTAATCAATTAAAGA

ATGATACGGAAAAAGTTACAAATCTGAAGGCACTTTTAGAAAAGTTGCCCAC

AATTAGTCAGTATGCTGCATTCTTACCTATCACACCTTCAGAGTTTAGATATA

TGAATTGTGAAATAAGCAGGGTGATTCTTCCAGAATTGTTATCCATTCATCTT

CCTTCATATCCGCCTTACTACCATGTAAGATATGAAGAAATTGGAGGTGTGGT

AAATAATATTCCACATCCAATTGCATAACATCTTTTTTTTAATCACATTATTTC

CAGGTATGCTTCTTTTATTGACGAAATGGATTCCAGTTCTATCTAGAAAGAGT

ACTTTTTTTCAATATTTACTTTGTTTATTCTTGATCACAATTATGAATAGTTTG

TTTTTCCGTCAACAGTTTGTGGTAGTTTTATCGCTGATATGTATTTTATTCTTA

CCATTTATTCTGTTTTTTGTAGAATATATATTTGTTGAGAGACAATGGAAAAA

GTTGCTTACTATTTATAAAAAAAATAAAATTTTTATCCAATCTATTGTATGGT

TTCCTGTTTTAGAAGAAATAATTTTCCGTTTTTTTATTTATCAATACTGTGAGT

TATTTGATTTCAGTAATATCCAGTATATATTACTAGCCACCTTTTCATTCGTGA

TTGCACATATTTTTTATCAAGGAGTGTCTTCAATTGTCAAAATACTATTTTCTT

TTATATTAAGTATATTATTTTTATTAACACTAAATATATTTTTGACAATAATAA

TTCACTGTATTTTCAACTTTTTAGTTTATATAGTTCGTACTAGTAAATATGAGA

ACCACCGTAATTGGTAAGCTGACAGTATTATATTAATTTAGCTGTTAAAGTAT

GGAAATACCAATAAATCAACGTTTTTCAACTTATGAAATGCTTGGTTCTCATT

AAAAATGATATATTTCCCTACCA

SEQ ID NO 4;Promoter PlIsA

GTGGAGTGAAATATAAGTTAGAGATTATTTTTCGATTAGGTGTACTTTTTTTTT

GTCTTCGTTTTATAGATTTTAGTATTTTTTTAATCTTGTATTATTAATGATAGTT

ATCTATAGCTTATATGCAGATTATGGAGTTGTATTAGTAAAAAAGCCCTGAA

GCCTCAGCATTATTGAGTTTCTAGGGCTTTTTTATGTCGAGTGATAACCGTTTT

TTATTATCGTACTATTTTTGTGGATAATATTTTGTCCAATTATACATATATACT

TTTGGAGATATTTAAACCAATTTTGCATATTATCAAACGGAGGGATATATTTT

TTATAAAAATAATTAAAAAAATTATTTAATTTTCTGAAATAAACAAAGAATTT

ATTTATTTTGATAATTATATTGAAAACGATTTCACAATGTGATAGGATGAACT

AAGGGATAATTTATTCCAAAAAATAAAAAGGAGGCATTTGA

SEQ ID NO 5; UsA

ATGAATATTAAATCACAATCATCAAATGGCTACAGTAATAATGCTGTAGGCT

CTGAAGCAATGAACTATGCAGCTGGATGTTGCTCATGTTCTTGTTCAACTTGC

ACATGTACATGTACATGCGCATCATCTGCTGCAACCAAAATGTAA

(Predicted leader peptide-encoding region in standard text, predicted unmodified leaderless propeptide-encoding region in bold text)

SEQ ID NO 6; Unmodified LIsA

MNIKSQS SNGYSNNAVGSEAMNY AAGCCSCSTCTCTCTCASSAATKM

(Predicted leader peptide in standard text, predicted unmodified leaderless propeptide in bold text)

SEQ ID NO 7; UsG

ATGAATAATATTTTGGAAACGAAAAATTTGAAAGTTACAATAAATAATAAAG

TAATTCTATATTTAGATAAAGAAGTTTGTATTTCAGAAAAAGACAAAGTTGCC

ATTCTAGGAGACAATGGAGCTGGGAAAACCACGTTAGTAAATAGTATTCTTG

GTGAAAAGAACTCTTCTGGAGAAATTACCAAAAAGTTTAAAAAAAATGACTG

TGGTGTAGTGTTTCAAGAAAATGCATATAATGATTTGATGAAAGTTTATGAAT

TGATTACTTTAGTTCTGCCACATCTAAAGAAAAAAGAGAGAGCGCAGTTTTT

ACACAAATATGAACTTGAAAGTTTGAGAAAAAAGTACATTAAAGACTTATCT

GGTGGAGAAAAACAACGACTAACACTATGTTTAGTACTAGAAAGTCATAAGA

AATTATATATCTTTGATGAGTTAACTTCAGGATTAGATTATAAGAAGAGACTA

GGTTTACTTGCTTTGATGAAAGAAAAAACTAAGGACGCGACGGTGATAAATA

TAACGCATTACTTTGAGGAAGTTGAAAACTGGGCAACGAAAGTTTTGATTCTT

CAAAAAGGAATTTTACTTTTTTGGGGAACAATATCAGAATTTTTCTCTAATTT

TCCTCATTATTCTGTTATTAAAGTAGATCAAATCGAACTAACAAAGATAGATG

AAACAGATATGACTTTTATGCAAAGCACAGATACAGGTGACGGGATAGCAGT

TATTTGTTCGGATTTGCAAATTCAGGAAGAGACAAAGAAAATTTTGGATAAA

AAAAACGTCACATACAACACAATAAAACAAAATATATATACAACTTATTTAG

TTGCATATTTGCGCGGAACATCAAGCAGTGAACAGGAGGTACTGATATAAT

SEQ ID NO 8; LIsG

MNNILETKNLKVTINNKVILYLDKEVCISEKDKVAILGDNGAGKTTLVNSILGEK

NSSGEITKKFKKNDCGWFQENAYNDLMKWELITLVLPHLKKKERAQFLHKYE

LESLRKKYIKDLSGGEKQRLTLCLVLESHKKLYIFDELTSGLDYKKRLGLLALMK

EKTKDATVINITHYFEEVENWATKVLILQKGILLFWGTISEFFSNFPHYSVIKVDQI

ELTKIDETDMTFMQSTDTGDGIAVICSDLQIQEETKKILDKKNVTYNTIKQNIYTT

YLVAYLRGTSSSEQEVLI

SEQ ID NO 9; HsH

ATGAGTTATTTATGGACAAGTATTAAAATGCAATTTAGAATTCCAGTATCTGT

GTTTTTCTCATTATTATTTCCACTAATCATGATGTTCGCTATGGTTACTTCTTA

TGGAAACTTTGATATTGGTGAAGGTTATCATTTTGTAGATAAATATTTTTTGA

TTAGTACAGGGATGGGCATGTTACCAATAGCATTAATCAGTTTTCCAATCTGG

CTTGGTGAAAGTGTTCAAAATAAAAGCTATAAAAGATTAGAATATTTTGGAT

TGAGTGCACAGAAAATAATTGTTTCAGATGTATGTTCTTATATTTTGCTAACA

GCCTTAAGTATTTTTGTCAATATACTGTTTGGCTACTTGGTATATGGACTACAT

ATCCCAGACTGGCAATATTTTATTGCTTATGTTTTGCAATGTCTGTATTGTAAT

CTAGTGCTTTTGATTTTTGGTGCCTTGCTTGCTTTAATTATTAAAAATCCCCGG

ATATTGATGCCAGTAGGAATGTGTTTATTATTTATGTTTTATATTTTTACAGGA

ACATTTTCATCTTTTTCGGAACTTCCTAAATCATTTCAAGCAGTTGGTAATTTT

TTACCTATGAAATATATAATGAATGATTTATTTAATGTTTGGACACAAAATAA

ATTGTTCATCTCTAAGTTTTTAGCTTTAAACACGTTATATGGAATCATCTTATC

ATTCGCACTAGTTGCTTTTTTGTTGAGGCAAAAGAAAATAAAAATTAAACCAT

AA

SEQ ID NO 10; LIsH

MSYLWTSIKMQFRIPVSVFFSLLFPLIMMFAMVTSYGNFDIGEGYHFVDKYFLIST GMGMLPIALISFPIWLGESVQNKSYKRLEYFGLSAQKWSDVCSYILLTALSIFVNI LFGYLVYGLHIPDWQYFIAYVLQCLYCNLVLLIFGALLALπKNPRILMPVGMCLL FMFYIFTGTFSSFSELPKSFQAVGNFLPMKYIMNDLFNVWTQNKLFISKFLALNTL YGIILSFALVAFLLRQKKIKIKP

SEQ ID NO 11; HsX

ATGAAAAAAAAATTTAGTAATCCCACGTTTAGAATTATTGCATCAATTGTTCT

AGGGATTTTGATAGGTGTTCTTATTTGCTTTGTGGCTATTGGTTTAGGTTACAT

TCACATGAATGATGGCACATTAAAAGAGTATAGTGTGAAAATTTTCGGACTA

ACTATTTTTGATATTAAAAGAGTTGGTAGTGAGATGGTGGGAACACCTAATA

ACACTAGCATGATGTTTATAGGGGTTATTATTTCTATGATACTAGCTATTGTT

GTAGAAATTATTGTTTCGTTAAAAAACAGACATAGAAAGGAAACAGCAAAAT

GA

SEQ ID NO 12; LIsX

MKKKFSNPTFRIIASIVLGILIGVLICFVAIGLGYIHMNDGTLKEYSVKIFGLTIFD IK

RVGSEMVGTPNNTSMMFIGVIISMILAIWEIIVSLKNRHRKETAK

SEQ ID NO 13; UsB

ATGATCGACTATGAGAAAAAAGGCTTTTTTAACATCCACACATTGGTAAATA

AAGATAATGCTAATATTTCTAATAGTGATAATAAACATATTTATTCCCAGCTG

ATGTCTGGTAATGGCAATTCACCAATGCTAGGTTATTTATTAAATATGAATAA

ACAAAACTTGAATGACTTCAAAAGTATCATGTTTTATAATGAATCTAATCTAG

CTTCTTTAATTAACGAGGCAAGAGAAATGGAAGAATTAATAGATAGCTCAAC

TCTTTTTTTAAGTAAAACAAATAAAAAAATTAAAACCCATTTTTCTAAAGTGC

TTGAGCAAAGACATAGTACAAGAAATTTT GTATACGAAACCATGGATTTAT C

AACATTCTCAAATATCATACAGTTTTCTTTTGGCCTTAGTACAAGAAAGTTGG

TTTATAACGACTTACAATCAACCACCAGGCATTATTCTTCTGGGGGCGGTTTA

TATCCAATTGATGTTTTTTTGTATATAAATAATATTTCTGGAATTGCCAAGGG

TATATATAAATATCAGCCATACACGCATAGCTTGCATCCTCTAGATGTAGATA

AAATTGATGTAGAGTCGTTTTTCGTAGGTGATAATATTGACACTTCTAATATG

AATTTTTGTGTTTTCTTTGGATACTCTATTAATAAAAACTATGTGAAATATGG

AGAACTATCTTTACTGAATACGTTTGTGGAACTAGGAGGAATATCACACAAC

TTTGATTTAGTTTGTCATTCGGTTCACTATACAAGTTGTCCTATTGCAGGTTTT

AACAAGTCGTACATAGAGAAGCTTTTATATTTAGATGGGATAAATGATCACA

TTATATTTACAAATATTTGTGGAAAGGAATGA

SEQ ID NO 14; LIsB

MIDYEKKGFFNIHTLVNKDNANISNSDNKHIYSQLMSGNGNSPMLGYLLNMNKQ

NLNDFKSIMFYNESNLASLINEAREMEELIDSSTLFLSKTNKKIKTHFSKVLEQRH

STRNFWETMDLSTFSNIIQFSFGLSTRKLVYNDLQSTTRHYSSGGGLYPID VFLYI

NNISGIAKGIYKYQPYTHSLHPLDVDKID VESFFVGDNIDTSNMNFCVFFGYSINK

NYVKYGELSLLNTFVELGGISHNFDLVCHSVHYTSCPIAGFNKSYIEKLLYLDGIN

DHIIFTNICGKE

SEQ ID NO 15; HsY

ATGAAAAATTTTGATATTAGAATAGGAACGCAGACAATTGACAATGATACGG

AATTCATTTTAAAAAGAGGGGTTATTCATAAGAATGAGATCGTTATCAATAA

AGAGGAAAGTTCAAAAGAATTTGTTTCAACATTTAAAGAATTGGTCAAGAAA

AAAACTATTACTATATCTTCAGAAGATGCAATCTACAATGATTTTGAAACACT

TACAAAATTTGGCTTTTTAACAATTTCTAAAAACCAAACCTTAAAACCTCTCT

TGATAGTTGAAGATGCTTTGTTTGATGACGTGAAGAGCTATTTCCAGGAAGA

AATTGAGATTTTATCATCCTCTGAATTTCTTTTAAAAAAAGATATTCGGTTAC

TTACTGAGAACAAAGATATATTGCAACTTACTAAACTAGTAGATGAAAAAAA

GGAATTAATGAAAAATTATAATTATATTTATTTAATCACAAACATTTCCAATA

TTTCCTTATTACGTGGATTTAACAAGTTAATGAAAGAGACTAACAGTGTTAAT

ACCATCGCATTTTTTGATAATGAAAATGTATTTGTTACTTGTATAGAACATGG

AGAAACTGGCTGCTATGAGTGTTTAGAGCGGCAACTACTCTCTCATTTTGATG

GTGTTGTTACGGATTATTTAGTACAGTCAGAAAATAATGTTTCCACAGCTGAA

TTGATGTTTGTTCTTTCAATCATAAAAAAAGAAATCGAAAATACCTCTATTTA

TGGTCAATCTTCTCTGTTAGGCAATCTTCTCCACTTTAATTTTAATAACTACGA

GTATACGTTTAATACAAATAGAATCCAAAGTTGTTGTTCTACCTGTGCTACTT

TCAATAATATATTATTTGAAGAGCAAAACATTCGGTCAGTGAATATACTAAA

GGAGCTGATGAGTAGTGATTAG

SEQ ID NO 16; LIsY

MKNFDIRIGTQTIDNDTEFILKRGVIHKNEIVINKEESSKEFVSTFKELVKKKTITI S

SEDAIYNDFETLTKFGFLTISKNQTLKPLLIVEDALFDDVKSYFQEEIEILSSSEFL L

KKDIRLLTENKDILQLTKLVDEKKELMKNYNYIYLITNISNISLLRGFNKLMKETN

SVNTIAFFDNENVFVTCIEHGETGCYECLERQLLSHFDGWTDYLVQSENNVSTA

ELMFVLSπKKEIENTSIYGQSSLLGNLLHFNFNNYEYTFNTNRIQSCCSTCATFNN I

LFEEQNIRSVNILKELMSSD

SEQ ID NO 17; HsD

GTGATTAGCATACAAAATAATTTGGAATATAACAAGTTACGTTGGGAAACTT

TGAGTGGAAATGTAACAGGAATATGGGAGAATAATAAGTTCTTTCTAGGCTC

TAGTTCTTACCCTATTATGAAGTATCATTACATTACAGCTAATTTTGTTAATTT

TGAAAAACATATTTCTGAAAATATGCCTAAAATAAGTTACCATTTAAGTGGA

TATGGTGTTAATTTTAATGAAGCGCTTGTTAGTTTTATTGGTGAAAGCGCAGA

GAGATATACATATTCCTTACTACCTACTATTATTAAGGACAGGATTATTTTTA

GATCATATGAAGAAATGACAAAAGAATATAAAACTGACTTAATATGTGAACT

TAAATACATAAATTCGTATTACTCTTCTGAAGTATGCGAAAATTATGTTACTC

CAAATGATACTATTCAATGGATAGCAATGAATTCTCTTGTTCATTCTGATAAA

AAAGTATGGATGCCTTTGCAATTTGTTACTATGTATACAGAAGAAATGTTTTC

TAATGAAAAAAGATATGTAACTAGCGCTGTATCAACAGGCACTGCTTGTCAT

GAAACTGTAGAAAAAAGTATAGAAAATGCCCTAATTGAATATCTTCAAATTG

ATTCCTTTAATTTATGGTGGTATGGAGGGTTTCGTGCGAGAGATATAGAAATA

GATATCACTCGAAATATATCAAGCTGGTTCGACAATCAAGTAGCTGTGAAAA

AATTTTTATCAAAGTTTAATGTACATTTTTCAGATATAAGTTTTGATAAATCA

ATTTATATTGTGCTATGCGAAATAGAGGCAAAAAATTCAAGTGATGCCTTTCC

TAAATATACTGTTGGCGTTCAAGGCGGATATTCACTAGATAAATCAATATATC

GTGCATTTATGGAATGTCTAACTGTACTAGAATATAATATGAATGTCACTTGG

ACAGATAAAGAGAAATTTCTTTCAGTCACACAGGAAACACGTGTTATAGATA

ATTTGGATGATAATGTTATTTATTATTCAAAGTATGGAAAACCAGAATTGCAA

TATAATACTAATCAATTAAAGAATGATACGGAAAAAGTTACAAATCTGAAGG

CACTTTTAGAAAAGTTGCCCACAATTAGTCAGTATGCTGCATTCTTACCTATC

ACACCTTCAGAGTTTAGATATATGAATTGTGAAATAAGCAGGGTGATTCTTCC

AGAATTGTTATCCATTCATCTTCCTTCATATCCGCCTTACTACCATGTAAGATA

TGAAGAAATTGGAGGTGTGGTAAATAATATTCCACATCCAATTGCATAA

SEQ ID NO 18; LIsD

MISIQNNLEYNKLRWETLSGNVTGIWENNKFFLGSSSYPIMKYHYITANFVNFEK HISENMPKISYHLSGYGVNFNEALVSFIGESAERYTYSLLPTπKDRπFRSYEEMTK EYKTDLICELKYINSYYSSEVCENYVTPNDTIQWIAMNSLVHSDKKVWMPLQFV TMYTEEMFSNEKRYVTSAVSTGTACHETVEKSIENALIEYLQIDSFNLWWYGGF RARDIEIDITRNISSWFDNQVAVKKFLSKFNVHFSDISFDKSIYIVLCEIE AKNSSDA

FPKYTVGVQGGYSLDKSIYRAFMECLTVLEYNMNVTWTDKEKFLSVTQETRVID

NLDDNVIYYSKYGKPELQYNTNQLKNDTEKVTNLKALLEKLPTISQYAAFLPITP

SEFRYMNCEISRVILPELLSIHLPSYPPYYHVRYEEIGGWNNIPHPIA

SEQ ID NO 19; UsP

TTGCATAACATCTTTTTTTTAATCACATTATTTCCAGGTATGCTTCTTTTATTG

ACGAAATGGATTCCAGTTCTATCTAGAAAGAGTACTTTTTTTCAATATTTACT

TTGTTTATTCTTGATCACAATTATGAATAGTTTGTTTTTCCGTCAACAGTTTGT

GGTAGTTTTATCGCTGATATGTATTTTATTCTTACCATTTATTCTGTTTTTTGTA

GAATATATATTTGTTGAGAGACAATGGAAAAAGTTGCTTACTATTTATAAAA

AAAATAAAATTTTTATCCAATCTATTGTATGGTTTCCTGTTTTAGAAGAAATA

ATTTTCCGTTTTTTTATTTATCAATACTGTGAGTTATTTGATTTCAGTAATATC

CAGTATATATTACTAGCCACCTTTTCATTCGTGATTGCACATATTTTTTATCAA

GGAGTGTCTTCAATTGTCAAAATACTATTTTCTTTTATATTAAGTATATTATTT

TTATTAACACTAAATATATTTTTGACAATAATAATTCACTGTATTTTCAACTTT

TTAGTTTATATAGTTCGTACTAGTAAATATGAGAACCACCGTAATTGGTAA

SEQ ID NO 20; LIsP

MHNIFFLITLFPGMLLLLTKWIPVLSRKSTFFQYLLCLFLITIMNSLFFRQQFVVVL SLICILFLPFILFFVEYIFVERQWKKLLTIYKKNKIFIQSIVWFPVLEEIIFRFFIYQYC ELFDFSNIQYILLATFSFVIAHIFYQGVSSIVKILFSFILSILFLLTLNIFLTIπHCIF NFL VYIVRTSKYENHRNW

The promoter, PlIsA, is located downstream of Imof2365 _1111 and it does not contain any of the motifs such as PrfA boxes or σ 8 binding sites, previously associated with virulence gene regulation in other L. monocytogenes strains. LIsA encodes the cytotoxin of the nucleic acid sequence of SEQ ID No. 1. The pathogenicity island shares homology with genes associated with the production of previously reported gram-positive bacterium produced cytotoxins, Streptolysin S, Staphylolysin S (Sts) and Botulysin S (Bts) In another aspect the invention provides the cytotoxin is Listeriolysin S (LIs). The cytotoxin has the following characters itcs:

(i) Haemolytic

(ii) Cytotoxic

(iii) Contributes to survival of the pathogen in polymorphonuclear neutrophils (PMN).

(iv) Contributes to the virulence of the pathogen as assessed by murine assays. The predicted mass of the leaderless peptide before modification is 2255.7. The peptide is active in a cell-associated form but when in cell-free culture supernatant it is inactive and requires re-activation by the addition of reagents. The re-activation reagent can be RNA core.

The invention further provides a cytotoxin comprising a fragment or a mutant of the cytotoxin of the invention. The invention further provides for probes or antibodies raised against the above-mentioned sequences.

In a further embodiment the invention provides a diagnostic agent to distinguish between high and low virulent strains of Z. monocytogenes, based on genes from the LIPI-3 pathogenicity island. The gene may be selected from the genes of thegene region flanked by two Rho-independent terminators, located after Imof2365_l 1 11 and HsP, respectively, and two Rho-independent duplicated glyoxalase-encoding genes {Imof2365_llll, 1121). The genes may be selected from the genes HsA, UsG, HsH, HsX, HsB, HsY, HsD and HsP. The invention also provides a diagnostic kit based on these genes. The agent or kit may comprise e.g. (i) Probes and oligonucleotides for PCR (both standard and real-time) and hybridisation or assays involving antibodies raised against the cytotoxin or other LIs proteins, (ii) blood agar based assays involving media-condition whereby LIs production is induced while hemolysin (LLO) is repressed (iii) Cell culture based assays etc , although they may form part of many other assays and kits.

The invention also provides for a pharmaceutical composition comprising the cytotoxin of the invention as a means of treatment of a disease by administering a pharmaceutically effective amount of the cytotoxin.

In a still further aspect the invention provides a disinfection agent comprising the cytotoxin defoned above.

The invention also provides the use of LIs in vaccine development. The invention also provides the use of the biosynthetic machinery (i.e. other LIs proteins) involved in the post-translational modification of LIs to post-translationally modify other peptides in a similar manner.

LIsA encodes a peptide, which the present inventors have designated Listeriolysin S (LIs). The 49 amino acid pre -propeptide encoded by UsA, resembles the unmodified SagA peptide through the presence of a leader region (26 amino acid) that terminates with a classical 'double glycine' bacteriocin-associated cleavage motif (AG) and a putative propeptide (23 aa) with a predominance of cysteine, serine and threonine residues (78%). While the way in which SagA becomes post-translationally modified, it is known that related peptides such as microcin B 17 (Mcb) and goadsporin (9, 10), a bacteriocin and a morphogenetic factor, respectively, are modified through the formation of thiazole and oxazole residues (or heterocycles) (11, 12). These modifications require the presence of an associated biosynthetic protein such as McbC (microcin B 17) or, most likely, GodE (goadsporin). LIsB shares homology with these proteins and is thus a member of the McbC-like oxidoreductases, which modify polypeptides by cyclizing thioesters to form rings. It is thus highly likely that LIs is also modified to incorporate heterocycle residues. Mutation of the corresponding Sis- associated gene sagB resulted in the elimination of haemo lytic activity (11). Based on study of the corresponding Group A Streptococcus pathogenicity island, it is highly likely that the majority (and possibly all) of the genes within LIPI-3 are required for LIs production (11). This theory is also supported by the observation that deletion of representative selection of the LIPI-3 genes (i.e. UsH, UsX, UsB, UsY and UsD) from a constitutive LIs producer (F2365LlsCδhly) results in the elimination of haemolytic activity. In contrast deletion of UsP from this background does not result in the elimination of haemolytic activity. It is unclear whether this is as a result of the non- involvement of UsP or because the need for HsP is somehow overcome as a consequence of expression from a strong constitutive promoter.

In addition to the predicted roles of LIsA and LIsB, as described above, the roles of a number of other LIPI-3 gene products can also be predicted on the basis of bioinformatic analysis. LIsGH represent the individual components of an ABC transporter (22.4% and 19.8% identical to SagGH, respectively). With respect to bacteriocin-like peptides, ABC transporters are most typically associated with export of the structural peptides or are involved in providing protection to the producing strain against the activities of the peptide generated (13, 14). Given that LIs does not exhibit antimicrobial activity, it is most likely that LIsGH is involved in peptide export. Mutation of the corresponding SIs-

associated gene sagG resulted in the elimination of haemo lytic activity (11) and we have established that deletion of UsH eliminates haemolytic activity from F2365LlsCδλ/y.

Given that both components of a two component ABC-transporter are required for functionality, it can be inferred that mutation of UsG would have a similarly detrimental impact.

The role of LIsD (24.7% identity with SagD) is unknown. It has been established that mutation of sagD results in the elimination of SIs activity {Datta, 2005 #370} . We have established that deletion of HsD also eliminates the haemolytic activity of

F2365LlsCδA/y.

LIsP has been annotated as an N-terminal protease. In a modified bacteriocin context, N- terminal proteases are most frequently associated with bacteriocin leader cleavage i.e. removal of the leader en route to the release of the active portion of the peptide (14).

Given their location within this region UsX and Us Y it was predicted that they too would play a key role but these proteins do not share homology with proteins of known function and thus it is not possible to predict their role by in-silico analysis alone. We have established that deletion of either of these eliminates the haemolytic activity of

F2365LlsCδA/y

Detailed Description of the Invention

The invention will now be described in greater detail with reference to the following figures:

Fig. 1. Arrangement of the Stapholysin S (sts), Listeriolysin S (Us), Streptolysin S (sag) and Botulysin (bts) associated genes in Staph, aureus ET3-1, L. monocytogenes F2365,

Strep, pyogenes MGAS 8232 and Cl. botulinum ATCC3502 respectively. Homology between the predicted 11s gene products and their sag and sts equivalents is indicated by a line and dashed line, respectively, with an associated % identity value. % identity values with respect to the predicted Sag and Bts gene products are also indicated. The amino acid sequence of the predicted unmodified structural peptides, StsA, LIsA, SagA and

BtsA is presented (boxed) with italicized letters indicating amino acids likely to constitute the leader region. Residues within the structural propeptide that are potentially modified are underlined.

Fig. 2. Comparison of the LIPI-3 -containing region (bottom) with the corresponding region of LIs " L. monocytogenes (middle), Z. innocua and Z. welshimeri (top). For Z. monocytognes strains, the strain name and serotype (in brackets) is presented and the designation of the first and last gene in each case is that designated in the corresponding genome sequence. Homologous genes (or in the case of the HsA-P, clusters of genes) are presented by matching colours.

Fig. 3. Induction of PlIsA by 13mM cumene hydroperoxide and 22 and 5OmM hydrogen peroxide. Data are presented as (A.) mean relative light units (RLU; photons s " ) ± standard deviations for three replicates, and (B.) one representative of three independent experiments is shown. The colour bar indicates bioluminescence signal intensity (in photons s "1 cm "2 ).

Fig. 4. Consequences of constitutive expression or deletion of Us genes. A. Haemolysis of Columbia blood agar (5% Sheep's blood) by F2365, F2365Ahly and F2365 AhIyAHsB when LIs is under the control of the natural PlIsA or constitutive PHELP promoters (F2365Lls c , F2365Lls c δ/?/y and F2365Lls c δ/?/yδ/kδ). In situations where haemolytic activity, the corresponding haemoltyic unit (H.U.) values are presented. B. Cytotoxicity relative to F2365 (100%) with respect to the J774, C2-Bbe and CT26 cell lines; F2365δhly - black, F2365LlsCδ/?/y - white. * - significantly different (P<0.05), ** - extremely significantly different (P<0.005) different. Error bars represent standard error of the mean.

Fig. 5. Comparison of the virulence of F2365 and F2365δ//s5. A. Levels (cfu - colony forming units) of wild-type and mutant F2365 in livers and spleens of Balb/C mice 3 days post-intraperitoneal infection. B. Survival in human PMNs after 2 hr. * - significantly different (P<0.05), ** - extremely significantly different (P<0.005) different. Error bars represent standard error of the mean. Materials and Methods Growth conditions

Listeria were grown in brain heart infusion (BHI) broth or agar (Oxoid) or Columbia blood agar plates (containing 0.5% sheep blood; LIP diagnostics, Galway, Ireland) at 37 0 C unless otherwise stated. Strains used are listed in tables 1 and 2. Escherichia coli EClOl (75) and TOPlO (Invitrogen) were used as intermediate vector hosts. Antibiotics

were incorporated as follows: Erythromycin (ery), 150μg/ml is. coli, 5μg/ml Z. monocytogenes; Chloramphenicol (cm), 10μg/ml is. coli and L. monocytogenes; Kanamycin (kan) 50μg/nύ E. coli, 25μg/ml Z. monocytogenes; Ampicillin (amp) 100μg/ml Z\ coli. S-bromo^-chloro-S-indolyl-b-D-galactopyranoside (X-GaI) was incorporated at a concentration of 40μg/ml. Strain variation studies

For strains of unknown lineage, lineage was determined by an allele-specific oligonucleotide PCR multiplex as described previously (5). LIP 1-3 status of strains was determined and confirmed through three distinct PCR reactions involving the primer pairs 11 18f-l 118r (all primers are listed 5' to 3'; TCTT ACCCT ATT ATGAAGT AT CA- TAACACCATATCCACTTAAATG), 1118degf-l 1118degr (GATATGTAACTAGCGC TGTATCAACNGGNACNGCT-CCCTCCATACCACCATAAATTAAAKGARTCDAT YTG) and UsAint-llsAsoeD (TGCAGCTGGATGTTGCTC - TAGCCCGGGCAGAACT AAAGT). Amplifications were performed in 50μl volumes with 150ng/μl concentrations of each primer, 2mM MgCb, 0.2 mM concentrations of each deoxynucleotide triphosphate, 0.5 U of Taq Polymerase, and 100 ng of genomic DNA. Amplifications consisted of 30 cycles of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C. Amplification products were resolved on 1.5% (wt/vol) agarose gel. Genomic Dissimilarity

Compositional bias of dinucleotide frequency analysis using the web-based application dcltarlio {http://dcltarho.amc.uva.nl). Deltarho calculates the genomic dissimilarity values d (the average dinucleotide relative abundance difference) between input sequences and the genome sequence of choice (16). A high genomic dissimilarity (d ) between an input sequence and the corresponding host genome sequence indicates a heterologous origin of the input sequence.

Investigation of PlIsA expression with a luciferase-based reporter system The promoter reporter vector pPLK2-lux was generated by amplifying the kanamycin resistance cassette from pTVl -OK (17) with the primer pair KanRF-KanRR (CCCTGCAGGTCGATAAACC-ACGAATTCCTCGTAGGCGC) and the introduction of the Pstl-EcoKI digested product into similarly-digested pPL2-lux (18). PlIsA was fused with the lux cassette within pPLK2-lux through amplification with PllsAfor-

PllsAbluntrev (ATTCGTCGACTTTTGATGCTTAAG-CATTCAAATGCCTCCTTTTT ATTT) and cloning into the Saϊl-Swal sites. The resultant plasmid was isolated from the intermediate host and introduced into F2365. Bioluminescence was investigated by washing overnight cultures and resuspending in an equivalent volume of spent BHI or BHI containing cumene hydrogen peroxide (13 mM) or hydrogen peroxide (22 or 50 mM) and quantified with a Xenogen IVIS 100 imager (Xenogen, Alameda, CA) with a 5 min exposure time. Constitutive expression of LIs

To place the Hs genes under the control of the strong constitutive synthetic promoter PHELP, PHELP DNA was amplified with the primer pair PHELPFsoe-PHELPRsoe (GTGGAGTGAAATATAAGTTAGAGG-TCGAGATCTGCAGATGATTGTGATTTA ATATTCATGGGTTTCACTCTC) from plasmid pPL21uxPhelp (19) and fused between two DNA fragments amplified from the regions flanking PlIsA with the primer pairs PllsAchangeA-PllsAchangeB (ACCTGCAGAAGGGGTTATTGA-CTCTAACTTATAT TTCACTCCAC) and PllsAchangeC-PllsAchangeD (AT GAAT ATT AAAT CACAATCA TC-TGGAATTCCCAGCTCCATTGTCTC) by SOE (splicing by overlap extension) PCR (20) and cloned into the RepA- shuttle vector pORI280 (21) in the intermediate RepA+ host, Escherichia coli EClOl (15). The resultant pORI280-help vector was introduced into F2365 already containing the RepA+, temperature sensitive helper plasmid pVE6007 (22). Successfully transformed cells appeared as blue colonies following plating on BHI-Ery-Xgal agar at 3O 0 C. To select for integrants, i.e. cells in which the pORI280-help vector had integrated into the F2365 genome by single crossover homologous recombination, F2365 pVE6007 pORI280-help was grown in BHI-Ery at 3O 0 C, subcultured twice (0.1% inoculum) in BHI-Ery at 42 0 C and streaked onto BHI-Ery-Xgal at 42 0 C. The introduction of PHELP upstream of HsA in Ery /Cm colonies was confirmed by PCR.

Deletion mutagenesis by double crossover homologous recombination The hly gene of F2365 was deleted through the amplification (using the primer pairs hlysoeA-soeB (TGGAATTCCACCTAATGGGAAAGT-GGGTTTCACTCTCCTTCT ACA) and hlysoeC-soeD (TGTAGAAGGAGAGTGAAACCCTAGTGTAGATAATCC- AAGCCCGGGACAACTAATCTGAC), splicing and cloning (into the temperature

sensitive shuttle vector pKSV7 (23)) of DNA flanking the gene followed by the introduction of the spliced product through double crossover homologous recombination as described previously (24). This strategy in combination with the primer pairs, UsBsoeA-soeB (ATTCTAGACAAGGTATAGAAAGGTTTGCTGTTTCCTTTCT- ATGTCTG) and UsBsoeC-soeD (C AGAC AT AGAAAGGAAAC AGC AAAC- AAATATTTGTG-TCTCC CGGGAAATAGCTCTTCAC) was also utilized to delete UsB from F2365 and F2365Lls c δ/?/y. Haemolytic assays

Haemolysis was assessed by spotting lOμl of overnight cultures onto Columbia blood agar plates and incubating for 24 hr at 37 0 C. Haemolytic titre assays were carried out as described by Ginsburg et al (Ginsburg et al. JEM 1965), with some minor modification. Washed Listeria were concentrated 10 fold i.e. to 2 x 10 10 cfu/ml and serially diluted (twofold) in a volume of 0.3ml of activation buffer (0.005M maltose, 0.00 IM MgS(V 7H 2 O, 0.001M cysteine) and incubated for 10 min at 37 0 C. ). 0.5ml of prewarmed sheep's red blood cells (0.2% in activation buffer) was added and the final volume adjusted to 1.0ml. The tubes were incubated at 37 0 C for 4 hr, centrifuged and haemolysis was assessed spectrophotometrically with a Softmax Pro spectrophotometer at 420 nm. Haemolytic units were calculated for each strain by taking the inverse of the last dilution to show complete haemolysis. Cytotoxicity assays

C2Bbel (CRC-2102; American Type Culture Collection) and J774 (ATCC TIB-67) cells were used for cytotoxicity assays. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter Glutamax (Gibco Laboratories, Grand Island, NY), 10% fetal bovine serum (Gibco), 1% (vol/vol) nonessential amino acids (Gibco), 1% (vol/vol) penicillin-streptomycin (Gibco), and 0.01 mg/ml human transferrin (Calbiochem) at 37°C in a 5% CO 2 atmosphere. For cell invasion assays, C2Bbel cells were trypsinized (Gibco), harvested by centrifugation at 400 x g for 8 min, and resuspended in 1 ml antibiotic-free DMEM containing 10% fetal bovine serum. Cells were seeded onto 24-well fiat-bottom tissue culture plates (Sarstedt, Leicester, United Kingdom) at a concentration of 3 x 10 cells per well. The plates were incubated for 72 h at 37°C in a 5% CO 2 atmosphere until confluence was reached. Overnight cultures of Z.

99

monocytogenes were washed and added at a multiplicity of infection of 100:1, and the plates were incubated at 37°C in a 5% CO 2 atmosphere for 6 hrs at which time cell-free supernatant was collected and cytoxicity assayed with the Cytotox 96 Non-Radioactive cytotoxicity assay (Pro mega, Madison, WI) according to the kit instructions. Murine virulence assay

Groups (N=5) of 15-week-old BALB/c mice were inoculated intraperitoneally with overnight cultures of F2365 or F2365δ/&5 resuspended in 0.2 ml of phosphate-buffered saline to a final concentration of 2 x 10 CFU/ml. Mice were sacrificed 3 days postinfection, and the numbers of Z. monocytogenes in the livers and spleens of infected animals were determined by plating serial 10-fold dilutions of organ homogenates on BHI agar. All procedures involving the use of animals were approved by the institutional animal care committee and complied with relevant legal guidelines. Isolation of human PMNs

Polymorphonuclear neutrophil granulocytes (PMNs) were isolated by a one step procedure based on a method described by English and Andersen (25). Briefly, 9 ml EDTA anticoagulated peripheral blood obtained from healthy donors was diluted with an equal volume of 0.9% NaCl and carefully overlain on a discontinuous double gradient formed by layering 4 ml of polysucrose/sodium diatrizoate adjusted to a density of 1.077 g/ml (Histopaque 1077) on 4 ml Histopaque 1119 in 15 ml conical centrifuge tubes (Starstedt UK). The tubes were subsequently centrifuged at 700 x g for 30min at room temperature. After centrifugation two distinct leukocyte cell layers (lymphocytes/monocytes and PMNs, respectively) were obtained above the bottom sediment of erythrocytes. The PMN layer was carefully aspirated and washed in DMEM supplemented with 10% fetal calf serum (DMEM-FCS). Centrifugation followed at 300 x g for 10 min at room temperature. After two further washes the cells were counted using trypan blue staining solution (Sigma-Aldrich Chemie, Deisenhofen, Germany) and the viability of the cells was confirmed to be above 97%.

Quantification of intracellular survival of L. monocytogenes in human PMNs. Human PMNs were isolated as described above and were adjusted to a final concentration of 1 x 10 6 cells/ml in DMEM-FCS. Overnight cultures of Z. monocytogenes were washed with PBS, and resuspended in DMEM-FCS at a final

concentration of 2 x 10 7 /mL. PMNs and Z. monocytogenes were combined to yield a final bacteria:PMN ratio of 20:1. Incubation was at 37 0 C in 5% CO 2 . After 30 mins the PMNs were washed, resuspended in fresh medium containing 50 μg/ml of gentamicin (Sigma) to kill extracellular bacteria, and further incubated until T=2hrs. Subsequently, the samples were washed with PBS, and cells were lysed by the addition of ice-cold water, serially diluted in PBS, and plated on BHI agar to determine the number of viable intracellular bacteria. Statistical analysis

In all cases the differences in mean values were analysed with an independent samples litest, following testing for conformity of data to assumptions of parametric statistics (Kolmogorov-Smirnov test for normality and Levene's test for equality of variances). Results

BLAST Sequence Comparisons

DNA and protein databases were screened, utilizing the NCBI sequence database, with respect to genes associated with the production of bacteriocins or bacteriocin like peptides with particular interest in sagB/SagB homologues. This revealed that lineage IL. monocytogenes strains, F2365 and H7858, possess genes that are homologous to SagB (24% identity). SagB is a protein associated with the formation of Streptolysin SIs. These homologues are situated within an 8-gene island, provisionally named LIPI-3 by the current inventors (Figure 1).

Bioinformatic analysis of LIPI-3 revealed the presence of several additional genes with homology to Streptolysin-associated genes (sag). The sag locus, which includes nine genes (sag A-sag I), is necessary and sufficient for the production of Streptolysin S. The sag genes consist of sagA (encoding a 73% Cys/Gly/Ser/Thr-rich structural propeptide with a bacteriocin-like leader), sagB (a putative modification protein containing a McbC- like oxidoreductase domain, cdO2142.1), sagE (a putative immunity protein), sagGHI (an ABC transporter potentially involved in export) as well as sagC, D and F (encoding proteins of unknown function).

The ORFs designated HsGHBDP encode LIsGH, both components of an ABC transporter (22.4% and 19.8% identical to SagGH, respectively), LIsB (24.7% identity with SagB), LIsD (24.7% with SagD) and LIsP has been annotated as an N-terminal protease

(frequently associated with bacteriocin leader cleavage). Given their location within this region HsX and HsY could also be of relevance. This is further illustrated in Fig 1. The Sis-like features of LIPI-3 include the presence of a previously un-annotated putative structural gene designated by the current inventors as HsA. LIsA is predicted to encode a peptide, which the present inventors have designated Listeriolysin S (LIs). The 49 amino acid pre -propeptide encoded by HsA, resembles the unmodified SagA peptide through the presence of a leader region (26 amino acid) that terminates with a classical 'double glycine' bacteriocin-associated cleavage motif (AG) and a putative propeptide (23 aa) with a predominance of cysteine, serine and threonine residues (78%) (Figure 1). LIPI-3 Detection

PCR was utilised to determine the strain variable nature of LIPI-3 (the 8 gene island between the 2 extreme rho -independent terminators). Listeria isolates representing a cross-section of the three lineages as well as non-Z. monocytogenes strains. A number of PCR primers were utilised which targeted different regions of the island. 1118f — 1118r and 1118degF-l 118degR both amplify a stretch within HsD while HsAint-llsAsoeD amplify from the middle of HsA into HsG.

LIPI-3 was not detected in any of the lineage II and lineage III L. monocytogenes strains tested or in any non L. monocytogeneous strains tested i.e. L. innocua, L.welshimeri, L. seeleri, L. ivanovii and L. grayi. 52% of lineage I strains tested were found to contain LIPI-3 (Table 1, Table 2). The LIPI-3 + strains corresponded to 13 of the 19 lineage I sequence types (STs) previously proposed, i.e. STs 3-10, 13-14 and 17-19. Furthermore, in addition to F2365 and H7858, responsible for epidemic outbreaks of listeriosis in California (1985) and the US (multistate; 1998-99), respectively, the LIPI-3 + set includes the listeriosis-outbreak strains from Halifax (1981), Lausanne (1987), Illinois (1994) and North Carolina (2000). In contrast, relatively few lineage I outbreak-associated strains (i.e. Massachusetts 1985, UK 1989) lack LIPI-3 + In Silico Analysis and Screening

In silico analysis established that in contrast to LIPI-I , which was present in a common listerial ancestor but then lost from non-pathogenic species (26), the very high genomic dissimilarity value of LIPI-3 (δ* of 0.1178) relative to that of the remainder of the F2365 genome (δ* of 0.0343) and its atypical %GC content (29.9 vs 38%; Table Sl) indicate

that it has been acquired relatively recently. Interestingly, this region of the listerial genome seems to be particularly variable in that H7858 possesses an additional 14 ORFs upstream of its Imof2365_llll equivalent and there is a large amount of inserted DNA (>17kB) at the corresponding location in EGDe (Fig. 2), both of which are absent from F6854, L. innocua CLIPl 1262 and L. welshimeri SLCC5334. Analysis of the corresponding regions from recently released whole genome shotgun sequence data of a number of Z. monocytogenes strains generated by the Broad Institute emphasises this point (27); Fig. 2. Such analysis also reveals that homologs of lmofl 365 _1111 and Imof2365_1120 are to be found in LIPI-3 strains, thereby confirming that the flanking rho-independent terminators mark the outer limits of LIPI-3 (Fig. 2). In silico screening also revealed that, in addition to specific L. monocytogenes, GAS, related group C and G streptococci Streptococcus iniae (28), other genome-sequenced Gram positive pathogens i.e. Staphylococcus aureus ET3-1 , a representative of the most abundant bovine mastitis- associated Staphylococcus aureus lineages, and a number of group I Clostridium botulinum strains, possess clusters of related open reading frames (ORFs). Although the existence of these islands has also been noted recently by others (29, 30), they have not been described in great detail. The putative products of these ORFs have been designated Staphylolysin S (Sts) and Botulysin S (Bts).

StsA encoded within this cluster is predicted to possess a leader (26 aa), leader cleavage site (AG), a structural propeptide (24 aa) with a large number of potentially modified residues (83% Cys/Ser/Thr) and an N-terminal CCSCSCS motif also present in LIsA (Figure 1).

Gene arrangement and % identity values established that the LIs (LIPI-3) and Sts clusters are more closely related to one another than to their SIs counterpart. In fact of the eight genes located on LIPI-3, only HsX does not have significant identity with its Sts equivalent. Notably however, within the Sts cluster, homology to both HsB and HsD is divided across two ORFs. Resequencing of this region has confirmed these to be genuine frameshifts.

The Bts gene cluster was found to closely resemble SIs gene cluster at a region predicted to encode a structural propeptide (33 aas) having up to 73% modified residues (Fig.l). Haemolytic assays

SIs is responsible for the haemo lytic activity of GAS. As a result the haemo lytic activity of LIP 1-3 was investigated to determine whether it was responsible for the production of a haemolytic factor. As all L. monocytogenes strains, regardless of lineage, produce the cytolysin listeriolysin O (encoded by My located within LIPI-I) (31-34) which could mask any LIs activity, a F2365Ahly mutant was created. The AhIy mutant was non- haemolytic when grown on Columbia blood agar. To determine whether the Hs genes are expressed under such conditions PlIsA was fused to a Lux reporter system in a promoter probe vector (pPLKm2) and integrated as a single copy into the F2365 genome. Realtime analysis with an IVISlOO imager (Xenogen) revealed that expression under routine laboratory growth conditions is negligible but that the promoter was strongly induced upon exposure to oxidative stress, including cumene hydrogen peroxide (13 mM) and hydrogen peroxide (22 and 50 mM) for 10 min (Fig. 3).

The induction is transient, dissipating gradually over a 60 min period. The transient nature of this induction, combined with the haemolytic nature of cumene hydrogen peroxide at these concentrations, restricted assessment of the consequences of expression of the LIs genes from a haemolytic perspective. To overcome this problem the Hs genes were placed under the control of a constitutive Iy strong synthetic Gram positive promoter PHELP in both F2365 and F2365Ahly such that the consequences of LIs production could be determined. Placing the genes under the control of P HELP enhanced the haemolytic activity of F2365 (F2365Lls ) and resulted in a haemolytic phenotype in the AhIy background (i.e. F2365Lls AhIy) (Fig. 4a). This haemolytic activity was eliminated when HsB was mutated in a non-polar fashion, confirming the link between the island and haemolysis (Fig. 4a). L. monocytogenes cells were employed for these, and other phenotypic assays, as it was apparent that LIs, like SIs, is active in a cell-associated form with no activity being apparent from untreated cell-free culture supernatant. Data analogous to that in fig 4c demonstrates that the constitutive LIs producing strain has activity against the mouse colon carcinoma cell line CT26. Cytotoxicity Assays

The cytotoxicity of the LIs gene cluster was also assessed using F2365Lls AhIy, F2365 AhIyAHsB and a number of different cell lines. F2365Lls AhIy was significantly

more cytotoxic than F2365Ahly against C2-Bbe (human enterocyte-like), J774 (mouse macrophage) and CT26 (mouse colon carcinoma) cell lines (Fig. 4b). Murine virulence studies

The contribution of LIPI-3 to pathogenicity was assessed by comparing the virulence of F2365 and F2365δ/&5 (Us ) following intraperitoneal inoculation of Balb/c mice. From these assays it was apparent that lls ~ possessed a reduced virulence potential as evidenced by significantly and extremely significantly reduced levels in the livers and spleens, respectively, relative to the corresponding F2365 infected mice (Fig. 5a). PMN Survival

A number of lines of evidence suggested that one of the roles of LIs may be to contribute to the survival of Z. monocytogenes in PMNs. Firstly, SIs contributes to the ability of GAS to withstand neutrophil killing (11); secondly, of the phagocytes, PMNs produce the greatest concentration of oxidative stress-inducing species; and thirdly, the initial response to intraperitoneal infection involves an influx of neutrophils). Notably, PMNs are essential for the resolution of Z. monocytogenes infections, playing a critical role in reducing the bacterial burden in the liver, spleen and central nervous system (35). When the intracellular survival of F2365 and lls ~ in purified human PMNs (human cells being used to establish the human relevance of the murine results) was compared after 2 hrs, it was apparent that wild-type F2365 again survived significantly better than the lls ~ mutant (Fig. 5b), confirming a role for LIs as a virulence factor mitigating survival in PMNs. Discussion

The current inventors have identified an island of genes, provisionally named LIPI-3, within the genome of a subset of strains of Z. monocytogenes. Regions within this island were found to be homologous to regions within the genome of Group A Streptococcus (GAS), S. aureus and Cl. Boutulinum. The regions of homology were those responsible for the biosynthesis of the highly cytotoxic Group A Streptococcus (GAS) virulence factor Streptolysin S, the S. aureus associated Staphylolysin S (Sts) and the Cl. Boutulinum associated Botulysin S (Bts). Previous studies have reported that Streptolysin S is responsible for the characteristic β-haemo lytic activity of GAS and plays a role in the survival of GAS in polymorphonuclear neutrophils (PMNs), cytotoxicity and inflammatory activation, contributing to necrosis and systemic spread.

It has been assumed throughout the literature that the thiol-activated cytotoxin listeriolysin O (LLO) was the sole haemolysin/cytolysin produced by L. monocytogenes strains. In the process of disputing this, the current inventors have established that LIP 1-3 is specifically associated with a subset of lineage I strains. To access the strain variable nature of LIPI-3, Listeria isolates representing a cross-section of the three lineages as well as non-monocytogenes species were tested. 52% of lineage I strains tested were found to contain LIPI-3. In addition to F2365 and H7858, responsible for epidemic outbreaks of listeriosis in California (1985) and the US (1998-1999) the LIPI-3+ set included listeriosis-outbreak strains from Halifax (1981), Lausanne (1987), Illinois (1994) and North Carolina (2000). No lineage π or lineage II strains tested or any of the non-monocytogenes strains tested were found to contain the island LIPI-3. LIPI-3 contains 8 genes including a previously unannotated gene HsA. LIsA encodes a cytolytic virulence factor designated Listeriolysin S. In the present study the current inventors have shown that Listeriolysin S has the following characteristics:

(i) Haemolytic

(ii) Cytotoxic

(iii) Contributes to survival of the pathogen in polymorphonuclear neutrophils

(PMN).

The predicted mass of the leaderless peptide before modification is 2255.7. The peptide is active in a cell-associated form but when in cell-free culture supernatant it is inactive and requires re-activation by the addition of reagents such as RNA core. These findings, coupled with the contribution of listeriolysin S at a critical junction during the pathogenic life of this pathogen, point to LIs as being a cytotoxin associated with the high virulence potential of lineage I strains.

Currently there are no efficient or reliable diagnostic tests available to distinguish between highly virulent and low virulent strains of L. monocytogenes . The current invention provides a solution to this by providing factor specific for a subset of lineage I strains that can be used in a diagnostic test as a marker of high virulence. Should it be accepted by regulatory agencies that LIPI-3 is indeed responsible for the apparent enhanced virulence potential of some strains of L. monocytogenes , it may be that the limits with respect to the quantities of L. monocytogenes that are permitted in foods will

be altered such that there are two limits. A low limit for the more virulent LIPI-3+ strains and a higher limit for LIPI-3- strains. Methods for the detection of LIPI-3 in

L. monocytogenes strains could involve conventional PCR, Real-Time PCR or DNA hybridizations.

Furthermore, listeriolysin S has the potential application to function as a chemo therapeutic agent, acting as an alternative to existing agents. The current study has shown that listeriolysin S is cytotoxic to a number of cell types including cancerous cells.

Table 1.

LIs status of non-lineage I Listeria

Lineage* Strain 1 Equivalent Original source Serotype LIs*

L. monocytogenes π A 33225a c LMB0455 3a 1 π A 33226a c LMB0456 3c 1 π B NCTC7973 Clinical l/2a 1 π B CD1061 Pork sausage non 4 1 π A 33022a c ATCC15313 Rabbit l/2a 1 π B DPC4605 SLCC2479 Unknown 3c 1 π B CD1198 ground turkey non 4 1 π B CD1038 Pork sausage non 4 1 π B CD1059 Pork sausage 1 2 1 π B CD241 Silage 3 1 π B CD1028 Pork sausage non-4 1 π B CD243 Silage 1 /2 1 π B CD1742 Pork sausage non-4 1 π A EGDe laboratory strain l/2a 1,2 π A 33234a F6854 turkey frankfurter l/2a 2 π A 10403S laboratory strain l/2a J π A J2818 turkey deli (US 1989) l/2a - 2 π A FSL N3-165 Soil l/2a J π A FSL Jl-IOl 33418, F6900 Clinical l/2a J π A LO28 laboratory strain l/2c - 2 π A FSL J2-003 l/2a 2 π A J0161 33419, FSL R2499 turkey deli (US 2000) l/2a J π A FSL F2-515 l/2a - 2 m A 33077a c 98-18140 bovine tissue 4b 1 m A 33115a c 93-500 Arabia oryx 4c 1 m B CD83 Silage 4 1 m A FSL J2 -071 4c J m A FSL Jl -208 animal clinical 4a J

L. innocua

CLIPl 1262 J

FH2333 Lettuce 1

FH2381 Sausage rolls 1

L. ivanovii

CD293 1

CD588 1

CD 165 1

L. grayi

ATCC25403 Corn stalks 1

ATCC25400 Corn stalks 1

FH2289 Cooked chicken 1

L. welshimeri

SLCC5334 2

FH2109 Chicken 1

FHl 968 Caesar salad 1

ATCC35897 Plant material

L. seelegeri

CD 166

FH2062 Cooked breakfast

CD944

Table 2

LIs status of lineage I L. monocytogenes

Lineage* Strain 1 Equivalent Source ST* Serotype LIs*

Clinical (Mass. outbreak,

I A 33013a c ScottA 1983) 1-l a 4b 1

Food (UK outbreak,

I A 33413a c Ts45 1988) 1-lb 4b 1

I A 33007a c RM2218 Food l-2a 4b 1

I A 33008a c RM2387 Food l-3a 4b + 1

I A 33083a c F1109 Food l-4a 4b + 1

Food (US outbreak, 1998-

I A 33420a NI-227, H7738 99) l-5a 4b + 1

Food (US outbreak, 1998-

I A 33233a c H7858 99) l-5b 4b +1,2

Clinial (US outbreak,

I A 33386a c NI-225, H7550 1998-99) l-5b 4b + 1

I A 33104a F2365 Ca 1985 l-6a 4b +1,2 π-1 19, TS43, Clinical (L.A. outbreak,

I A 33410a F.4565 1985) l-6a 4b + 1

FSL N3-008, Food (Halifax outbreak,

I A 3341 1a TS50, L.4760 1981) l-6b 4b + 1

FSL N3-022, Food (Switzerland

I A 33415a TS21, L-4486j outbreak, 1987) l-6c 4b + 1

I A 33120a ATCC19118 Animal l-6c 4e + 1

I A 33116a ATCC19117 Animal l-7a 4d + 1

I A 33015a c 12375 l-8a 4b + 1

Clinical (Illinois

I A 33424a FSL R2-503 outbreak, 1994) l-9a l/2b + 2

I A 33423a c G6003 Food l-9a l/2b + 1

I A 33068a c 8058 Animal 1-lOa l/2b + 1

I A 33038a c OB001385 Food 1-l l a l/2b 1

I A 33037a c OBOOl 350 Food l-12a l/2b 1

I A 33126a c 7034 Animal l-13a l/2b + 1

I A 33176a c 20240-954 Animal l-14a l/2b + 1

I A 33090a c 7675 Animal l-15a l/2b 1

I A 33028a c OBOOl 102 Food l-16a l/2b 1

I A 33390a FSL J2-064 l-16f l/2b 2

I A 33032a c OBOOl 186 Food l-17a l/2b + 1

I A 33186a c 20674-01 Animal l-18a l/2b + 1

Food (Nrth Car. outbreak, 4b but l/2b

I A 33421 C JO 144 2000) l-19a complex + 1

CD1 121 ground beef nd 4b

Clinical (Mass. outbreak,

F5817 1983) nd non 4 CD2088 pork sausage nd 4b

Clinial outbreak (Mass.

FSL Jl -220 outbreak, 1983) nd l/2b

DPC4608 SLCC 1694 Unknown nd non-4 1

CD1032 Pork sausage nd 4a 1

CD 147 Dairy enrichment nd non 4 + 1

CD1066 Pork sausage nd non-4 1

CD749 Ground beef nd non-4 1

CD748 Ground beef nd 4 1

CD246 Silage nd 4 1

CD878 Clinical nd 1 2

CD1078 Chicken nd l/2b

FSL J1 -175 Water nd l/2b

FSL J1 -194 Sporadic clinical nd 4b +

FSLN1-017 Trout in brine nd 4b

HPB2262 Febrile cases (Italy) nd 4b

Table 3

Island 1000 x δ* island 1000 x δ* genome % GC % GC

(genomic disimilarity) average Island Genome

LIs 117.8 34.3 29.9 38

Sts 61 .3 45 26.6 32.8

Sag 35.4 36.2 35 38.5

Bts 33.8 33.6 26.2 28.2

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