Use of novel virulence-specific genes as targets for diagnosis and potential control of virulent strains of listeria monocytogenes

Abstract

A method for identifying virulent strains of L. monocytogenes that includes the use of primers or probes in a PCR assay or hybridization technique that employs primers or probes, which are specific for virulence-specific genes of L. monocytogenes. Also provided is a method of control of L. monocytogenes strains that have been identified using the method of the present invention.

Description

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention involves the use of novel virulence-specific genes of Listeria monocytogenes as targets for specific diagnosis and potential control of virulent strains of L. monocytogenes. More particularly, this invention provides a PCR or hybridization method, which uses specific primers or probes corresponding to virulence-specific genes for the identification and control of virulent strains of Listeria monocytogenes.

[0005] 2. Background of the Technology

[0006] L. monocytogenes is an important cause of human food borne diseases world wide. A notable feature of L. monocytogenes is that it shows considerable variation in its ability to produce listeriosis. On the one extreme, some L. monocytogenes strains are virulent and can result in severe disease and mortality. On the other, some have limited capability to establish in the host and are relatively avirulent and harmless. Because manufactured food products detected with L. monocytogenes are recalled or downgraded (i.e., used for pet food), contamination with this species may render significant economic losses. With outbreaks of listeriosis due to contaminated foods on the increase in recent years, L. monocytogenes has become a major concern to the food industry and health regulation authority.

[0007] Apart from adapting stringent quality control measures during food processing procedures, frequent monitoring with specific laboratory tests for virulent strains of L. monocytogenes is vital in reducing unnecessary food product recalls and allaying consumer concerns. The current diagnostic methods are incapable of distinguishing virulent from avirulent strains of L. monocytogenes.

[0008] The complete genome of Listeria monocytogenes EGDe strain was reported recently (Glaser et al., 2001). Although this publication contains a list of all known and putative genes in L. monocytogenes EGD strain as well as their nucleotide sequences, it does not provide any information on the actual application of these genes. Therefore, although the DNA sequences of the genes described in this invention have been published and are in public domain through the release of the L. monocytogenes EGDe genome sequence, there are no prior publications on the functions of these genes or on their use for research or diagnostic purposes.

[0009] Previous research used PCR and DNA sequencing or restriction fragment length polymorphism of the L. monocytogenes hlyA, actA, and inlA genes to group L. monocytogenes into three genetic lineages, with the various lineages varying in potential for human virulence (Norton et al., 2001; Wiedmann et al., 1997). Ribotyping (sequencing of rRNA genes) was also used in this research. These assays are different from the present assay employed by the inventors in that they require either DNA sequencing or restriction digests following PCR amplification, while the present assay is simply a PCR assay. In addition, the hlyA, actA, and inlA genes are present in all L. monocytogenes isolates, while the virulence-specific genes described by the inventors are found only in virulent strains of L. monocytogenes.

[0010] Another PCR assay, random amplification of polymorphic DNA (RAPD) PCR, has been used to classify L. monocytogenes into genetic groups that tend to predict virulence. This technique is based on the use of nonspecific primers that bind to unknown sequences in the L. monocytogenes chromosome (Franciosa et al., 2001). The PCR assay employed by the inventors is based on primers that bind to specific virulence associated chromosomal sequences that we have identified.

[0011] Other assays have been described for differentiation of virulent and avirulent L. monocytogenes isolates. The “gold standard” for virulence testing of L. monocytogenes isolates is the mouse virulence test. This test is expensive, labor intensive, requires several weeks to complete, and requires regulatory approval to ensure humane treatment of animals. Assays have been described based on cell culture models; one correlated L. monocytogenes virulence with the ability of isolates to form plaques on HT-29 cells (Roche et al., 2001), and another correlated virulence with the ability to cause cytopathogenic effects in Caco-2 cells (Pine et al., 1991). Although the use of cell culture models represents an improvement over mouse virulence testing, it is still time-consuming and labor intensive.

[0012] Research has been published on the use of phenotypic detection of virulence factor expression (listeriolysin, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C) to separate virulent from avirulent L. monocytogenes isolates (Erdenlig et al., 2000). Research has also been published on the use of monoclonal antibodies for detection of virulence factor expression (listeriolysin and phosphatidylcholine phospholipase C) to distinguish virulent and avirulent isolates (Erdenlig et al., 1999). The dot blot hybridization technique described in this invention has also been previously published. For example, this technique was employed to identify virulence and avirulence associated markers of Dichelobacter nodosus—the ovine footrot pathogen (Liu & Yong, 1993). Several PCR assays have been described for species specific detection of L. monocytogenes (examples include Aznar & Alarcon, 2002; Bassler et al., 1995; Blais et al., 1997; Klein & Juneja, 1997; Norton & Batt, 1999; Winters et al., 1999). PCR assays for distinguishing all six Listeria species can be based on the 16S and 23S rRNA genes (Sallen et al., 1996) or the intergenic spacer region of 16S and 23S rRNA genes (Graham et al., 1997), or the iap gene (Bubert, et al., 1999). However, none of these PCR assays distinguish virulent L. monocytogenes isolates from avirulent isolates.

SUMMARY OF THE INVENTION

[0013] This invention involves the use of virulence-specific genes of Listeria monocytogenes as targets for specific diagnosis and potential control of virulent strains of L. monocytogenes and overcomes the above identified shortcomings of conventional detection methods.

[0014] Using a comparative screening strategy, the inventors isolated two potential virulence-specific clones from a recombinant DNA library from L. monocytogenes strain EGD. Specifically, a hybridization technique was used to compare genomic DNA from virulent and avirulent L. monocytogenes isolates to identify clones containing genetic markers that are uniquely present in either virulent and/or avirulent strains. DNA sequence analysis of the two virulence specific clones revealed that they contain gene markers that are distinct from the previously reported virulence gene cluster encompassing prƒA, plcA, hlyA, mpl, actA, and plcB. By employing primers derived from these as well as other newly identified virulence-specific gene markers, the inventors discovered a method by which virulent strains of L. monocytogenes can now be readily distinguished from avirulent strains through the formation of specific PCR products.

[0015] The method of the present invention for separation of virulent and avirulent L. monocytogenes isolates can be used to provide a scientific basis for the determination of when and if food safety recalls should occur when L. monocytogenes is isolated from food products.

[0016] In one embodiment of this invention, virulence-specific genes of Listeria monocytogenes are used as targets for specific diagnosis and potential control of virulent strains of L. monocytogenes.

[0017] In another embodiment of this invention, one or more of L. monocytogenes virulence-specific genes are used to detect virulent strains of L. monocytogenes.

[0018] In another embodiment of this invention, one or more of L. monocytogenes virulence-specific genes are used to detect virulent strains of L. monocytogenes by polymerase chain reaction (PCR) using primers specific for the DNA sequence from the gene(s) or by hybridization using a probe specific for the DNA sequence from the gene(s).

[0019] In another embodiment of this invention, the one or more L. monocytogenes virulence-specific genes are selected from the group consisting of: lmo0833, lmo2672, lmo1116, and lmo1134 (encoding putative transcriptional regulators); lmo0834 and lmo1188 (encoding proteins with unknown function); and lmo0333, lmo2470, and lmo2821 (encoding proteins similar to internalins).

[0020] In another embodiment of this invention, a combination of two or more of L. monocytogenes virulence-specific genes are used to detect virulent strains of L. monocytogenes by multiplex polymerase chain reaction (PCR) or hybridization using primers or probes specific for the DNA sequences from the gene(s).

[0021] In another embodiment of this invention, one or more of L. monocytogenes virulence-specific genes are used to detect virulent strains of L. monocytogenes by multiplex polymerase chain reaction (PCR) or hybridization using primers or probes specific for the DNA sequence from the gene(s) in combination with Listeria genus-specific primers or probes and/or L. monocytogenes species-specific primers or probes.

[0022] In another embodiment of this invention, the one or more L. monocytogenes virulence-specific genes are one or more genes that indicate virulent forms of L. monocytogenes or combinations thereof.

[0023] In another embodiment of this invention, the L. monocytogenes virulence-specific genes or their derivatives are used in the inhibition of growth, reduction of pathogenicity, treatment, or prevention of virulent strains of Listeria monocytogenes.

[0024] In another embodiment of this invention, virulent strains of Listeria monocytogenes are detected by amplification of L. monocytogenes virulence-specific genes from mRNA by reverse transcription-PCR (RT-PCR).

[0025] In another embodiment of this invention, virulent strains of Listeria monocytogenes are detected by one or more methods for detection of protein product(s) from L. monocytogenes virulence-specific genes.

[0026] In another embodiment of this invention, virulent strains of Listeria monocytogenes are detected by one or more methods for detection of protein product(s) from L. monocytogenes virulence-specific genes using either polyacrylamide gel electrophoresis, high-performance liquid chromatography (HPLC), mass spectrometry, or antibody detection methods (examples include immunofluorescent antibodies (IFA), enzyme-linked immunosorbent assay (ELISA), or Western blotting).

[0027] In another embodiment of this invention, virulent strains of Listeria monocytogenes are detected by one or more methods for detection of protein product(s) from L. monocytogenes virulence-specific genes by use of assay(s) specific for the function(s) of the protein product(s).

[0028] In another embodiment of this invention, the virulence-specific L. monocytogenes genes are used as a treatment strategy such that pharmaceutically active agent(s) would inactivate or alter the function of one or more of the proteins encoded by the virulence-specific L. monocytogenes genes, which would either kill the virulent L. monocytogenes or render it susceptible to the host immune system.

[0029] In another embodiment of this invention, one or more of the L. monocytogenes genes or promoter(s) for one or more of the virulence-specific L. monocytogenes genes is altered such that expression of the encoded protein(s) would be completely disrupted or altered. The said alteration or disruption of expression would render L. monocytogenes avirulent and effective as a live attenuated vaccine.

[0030] In another embodiment of this invention, the L. monocytogenes virulence-specific genes are selected from the group consisting of: lmo0833, lmo1188, lmo0834, lmo1116, lmo2672, lmo1134, lmo1333, lmo2470, and lmo2821.

[0031] In another embodiment of this invention, the one or more L. monocytogenes virulence-specific genes are one or more genes that indicate one or more virulent forms of L. monocytogenes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the DNA sequences of SEQ ID NOS.: 1-9 for each of the virulence-specific genes of Listeria monocytogenes according to the present invention.

[0033] FIG. 2 shows the DNA sequences of SEQ ID NOS.: 28-33 for each of the Listeria species-specific gene sequences according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Listeria monocytogenes is a small gram-positive coccobacillus that tends to form short chains of three to five bacteria. Infections from this pathogen occur worldwide in various animals and man (Gray and Killinger, 1966) and can be fatal in immunocompromised individuals such the elderly, pregnant women, newborns, diabetics and transplantation patients (Gellin and Broome, 1989). L. monocytogenes is of particular concern to the food industry and public health regulatory agencies because it can grow at refrigerator temperatures and because it is ubiquitous in nature (Farber and Speirs, 1987, Lamont et al., 1988). It has been found in a variety of foods such as vegetables (Heisick et al., 1989), milk (Donnelly and Baigent, 1986, Doyle et al., 1987), various cheeses (Rodler and Korbler, 1989), meat products (Farber et al., 1989), poultry (Carpenter and Harrison, 1989), and fish (Lennon et al., 1984; Erdenlig et al., 1999). Of the 13 known serotypes of L. monocytogenes, many of which are found in foods, only three serotypes (1/2a, 1/2b, 4b) are associated with the majority of human illness (Schuchat et al., 1991). However, not all strains of these L. monocytogenes serotypes are pathogenic, with some strains having either no or low-level virulence (Hof and Rocourt, 1992). Previous work at the College of Veterinary Medicine at Mississippi State University indicated that L. monocytogenes isolates from channel catfish vary in virulence using the mouse model, with some isolates being highly virulent and others being completely avirulent (Erdenlig et al., 2000). There is also molecular evidence for the existence of genetic lineages of L. monocytogenes that vary in virulence (Norton et al., 2001; Wiedmann et al., 1997). This data indicates that food safety recalls based solely on detection of L. monocytogenes without determination of virulence could lead to unnecessary recalls, which would have devastating consequences on food producers and processors. To prevent economic losses due to food recalls, and to reduce human food safety concerns, it is important to understand what causes certain L. monocytogenes to be virulent and to devise ways to accurately ascertain virulence.

[0035] L. monocytogenes is a facultative intracellular pathogen, and some of its best-known virulence factors contribute to its ability to survive inside professional phagocytic cells. After it is phagocytosed, L. monocytogenes lyses the host vacuole and escapes into the cell cytoplasm. This step is mediated by listeriolysin (LLO) and phosphatidylinositol phospholipase C (PI-PLC) (Camilli et al., 1993, Portnoy et al., 1988). The bacteria are then propelled through the host cell cytoplasm by inducing the polymerization of host actin, a process that is mediated by a surface protein designated ActA (Domann et al., 1992). The bacteria then apparently spread from cell to cell by inducing formation of pseudopod-like structures containing bacteria that are internalized by neighboring cells. A second phospholipase, phosphatidylcholine phospholipase C (PC-PLC) is required for this step (Vazquez-Boland et al., 1992). A zinc metalloprotease, Mpl, may be required for activation of PC-PLC (Poyart et al., 1993).

[0036] The genes encoding these virulence factors are clustered on the L. monocytogenes chromosome between the ldh and prs operons: prfA (PrƒA, regulatory gene), plcA (PI-PLC), hlyA (LLO), mpl (Mpl), actA (ActA), and plcB (PC-PLC) (Portnoy et al., 1992). This gene cluster is one of the most well studied regions of the L. monocytogenes chromosome; there have been numerous publications on the roles that these genes play in virulence (Bohne et al., 1996, Bubert et al., 1999, Freitag and Jacobs, 1999, Kuhn and Goebel, 1995, Smith et al., 1995).

[0037] Previous work at the College of Veterinary Medicine at Mississippi State University has shown that expression of LLO and PC-PLC is valuable in indicating the pathogenicity of L. monocytogenes isolates (Erdenlig et al., 1999; Erdenlig et al., 2000). Expression of LLO and PC-PLC in seven L. monocytogenes isolates were compared, four of which were virulent in mice and three of which were avirulent in mice. Expression of both LLO and PC-PLC was present in all four virulent strains, and expression of LLO and PC-PLC was absent in two out of three avirulent strains (Table 1). None of the three avirulent strains expressed both LLO and PC-PLC.

TABLE 1
Application of mAbs to detect the presence of
virulence factors from L. monocytogenes channel
catfish isolates and their correlation to pathogenicity
L. monocytogenes
catfish isolate	Serovar	LLO	PC-PLC	Pathogenicity1
ATCC 15313	1	−	+	−
ATCC 19115	4b	+	+	+
EGD	½a	+	+	+
CCF 12	1	+	+	+
HCC 72	1	+3	+	+
HCC23	4	+	−	−
1Pathogenicity data for CCF 1, CCF 4, HCC 7, and HCC 23 are published in Erdenlig et al. (1999).
2CCF = channel catfish fillet; HCC = healthy channel catfish organs.
3HCC 7 is weakly positive for LLO.

[0038] DNA sequencing of the promoters from the virulence gene clusters of these seven L. monocytogenes isolates were completed. The promoters that were sequenced control expression of the hlyA, plcA, prƒA, and plcB genes. In addition, the entire prƒA gene was sequenced from the seven isolates because PrfA binds to each of these promoters to control transcription. The sequences were obtained by first amplifying the regions of interest by PCR and directly sequencing the PCR products.

[0039] The sequencing results provide evidence that there are distinct genetic lineages of L. monocytogenes based on the virulence gene promoter sequences. Phylogenetic analysis indicated that in three of the promoters, the seven strains grouped consistently into three genetic lineages. In the fourth promoter controlling hly (LLO) expression, five out of seven isolates were grouped into the same genetic lineages. The different groupings of the other two strains at this promoter possibly reflect differences in expression of LLO.

[0040] The sequencing results also revealed potential sequence differences that could explain the differential expression of LLO and PC-PLC between isolates. In one isolate that fails to express PC-PLC, two amino acid substitutions were detected in PrfA. In the hly promoter, there were three nucleotide substitutions in the strain that fails to produce LLO compared to other strains. In one of the plcB promoters, there were four nucleotide substitutions in the promoter region of a non-PC-PLC producing strain compared to other strains.

[0041] However, the sequencing results also demonstrated that these genes (prƒA, plcA, hlyA, and plcB) are not good candidates for the development of PCR-based tests for distinguishing virulent from avirulent strains. These genes are present in all L. monocytogenes isolates (and even some other Listeria species), and the sequencing results demonstrated that the sequence variations in these genes between virulent and avirulent isolates are too few to allow development of PCR primers that would reliably distinguish virulent and avirulent isolates.

[0042] Therefore, the goal was to identify other gene markers that could be used for distinguishing virulent L. monocytogenes isolates from avirulent isolates. Although the genome sequence of virulent L. monocytogenes strain EGD recently became available (Glaser et al., 2001), the sequence of avirulent L. monocytogenes isolates are not available for comparison to identify these unique genes. Therefore, dot blot hybridization was used to identify L. monocytogenes virulence-associated markers, which is a technique that had been previously used to detect chromosomal markers that are unique to both virulent and avirulent isolates of Dichelobacter nodosus, the causative agent of ovine footrot (Liu and Yong, 1993). These markers identified from D. nodosus were used as the basis for development of a diagnostic test that can be used to differentiate virulent, intermediate, and avirulent isolates of this species (Liu, 1994).

[0043] To prepare for dot blot hybridization, genomic DNA was prepared from the known virulent and avirulent strains of Listeria monocytogenes using a standard protocol (Ausubel et al., 1994) and suspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The purified DNA from virulent strain EGD and avirulent strain HCC23 was partially digested with restriction endonuclease Sau3A I. Digested DNA was separated by agarose gel electrophoresis, and fragments in the 0.5-3 kilobase range were excised and eluted. The size fractionated DNA was then cloned into BamH I digested plasmid vector (pGEM-3Z; Promega). The resultant recombinant DNA libraries were transformed into E. coli XL1-Blue MRF, and clones with insert were identified by blue-white screening. Plasmid DNA was isolated from individual clones in batches of 50 using a rapid alkaline lysis procedure. Inserts were isolated by digestion with Pst I and EcoR I, separated from vector DNA by agarose gel electrophoresis, eluted by the phenol-thaw method, and labeled for hybridization using the ECL protocol for labeling double stranded DNA (Amersham Pharmacia Biotech). If inserts contained Pst I or EcoR I restriction sites, inserts were recovered by digestion with Sma I and Hind III.

[0044] The dot blot hybridization was conducted using the procedure described by Liu and Yong (Liu and Yong, 1993). Briefly, DNA from each of the four virulent strains and the three avirulent strains were heated at 100° C. for 3 minutes before being mixed with an equal volume of 1.8 M NaCl, 0.18 M sodium citrate and 4.4 M formaldehyde. Fifty microliters of DNA from each of the seven strains (0.5 g DNA/dot) was spotted onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech) using a dot blot apparatus (Schleicher and Schuell). DNA was spotted in 50 panels, with each panel containing one dot from each of the seven strains, and fixed on membranes using UV light in a Stratalinker 2400 (Stratagene). Dot blot panels were separated from each other and individually hybridized with the labeled inserts.

[0045] Inserts were identified from these hybridizations that demonstrate preferential binding to virulent or avirulent strains. Inserts from identified clones were sequenced on both ends using primers from the vector sequence. Clones from the virulent strain EGD were easily identified based on the available genome sequence data, but inserts from the avirulent strain required sequencing the entire insert using a primer walking strategy. Southern hybridizations using labeled probes from the identified clones were conducted using genomic DNA from all seven strains to confirm results from the dot blot hybridizations.

[0046] Through this comparative screening procedure, two recombinant clones (Lmo2-28 and Lmo2-432) were identified from the genomic DNA libraries of L. monocytogenes strain EGD (NCTC7973). Following nucleotide sequence analysis of these two clones and subsequent BLAST searches at GenBank, clone Lmo2-28 was found to contain parts of lmo0833lmo0834 of L. monocytogenes EGDe, which encode a putative transcriptional regulator and an unknown protein. Clone Lmo2-432 was found to contain part of lmo1188 of L. monocytogenes EGDe, which encodes an unknown protein. Because of this interesting finding and the fact that transcriptional regulators are specialized DNA binding proteins that play essential roles in the regulation of RNA synthesis and gene expression within bacteria, attention was focused on genes encoding transcriptional regulators in L. monocytogenes. As a result, several other genes (lmo2672, lmo1116, and lmo1134) were selected from the list of L. monocytogenes EGDe genes (Glaser et al., 2001) for further evaluation (Table 2). Furthermore, because internalins are found exclusively in Listeria, additional attention was also directed to L. monocytogenes EGDe genes that encode putative internalins. Thus, the inventors selected three genes (lmo0333, lmo2470, and lmo2821) that code for proteins similar to internalins for assessment. The Listeria monocytogenes virulence-specific genes used as examples of the present invention are listed in Table 2. Sequence lists for each of these genes are shown in FIG. 1 as: lmo0833 (SEQ ID NO: 1), lmo1188 (SEQ ID NO: 2), lmo0834 (SEQ ID NO: 3), lmo1116 (SEQ ID NO: 4), lmo2672 (SEQ ID NO: 5), lmo1134 (SEQ ID NO: 6), lmo0333 (SEQ ID NO: 7), lmo2470 (SEQ ID NO: 8), and lmo2821 (SEQ ID NO: 9). Primers [forward primers (5′-3′) and reverse primer (3′-5′)], corresponding to each of the L. monocytogenes virulence-specific genes are also shown in Table 2. As indicated in the Table, these primers are sequentially designated as SEQ ID NOS.: 10-27. The oligonucleotide primers, which were designed from each of these genes were assessed in PCR against a collection of 29 L. monocytogenes strains (Table 3).

TABLE 2
Identities of novel L. monocytogenes virulence specific gene markers
							PCR
	Genome	Putative	Size	Forward primer	Reverse primer	Primer	product
Gene	location	function	(aa)	(5′-3′)	(5′-3′)	positions	(bp)
lmo0833	223780-	Transcrip-	296	ggctattctttagcggagga	agtagcgcgagggatttgta	223996-224015;	638
	224730	tional		SEQ ID NO. 10	SEQ ID NO. 11	224633-224613
		regulator
lmo1188	53621-	Unknown	483	tttcgccgttagaaaatacga	ttcggacaaaaatttgaatgg	54027-54047;	663
	55085	protein		SEQ ID NO. 12	SEQ ID NO. 13	54689-54668
lmo0834	224810-	Unknown	237	aacttcgcatttgttatgtgttac	tcactgaccattcctccaaa	224940-224963;	594
	225537	protein		SEQ ID NO. 14	SEQ ID NO. 15	225533-225513
lmol1116	262997-	Transcrip-	257	gggaacgatgaaaacgaaga	tggcttatcgcacaagctaat	263006-63025;	591
	263783	tional		SEQ ID NO. 16	SEQ ID NO. 17	263593-263573
		regulator
lmo2672	25985-	Transcrip-	268	cggcacacttggattctcat	agggctagtgacggatgcta	26117-26136;	481
	26804	tion		SEQ ID NO. 18	SEQ ID NO. 19	26597-26578
		regulator
lmo1134	8009-	Transcrip-	115	acccgatagcaaggaggaac	aacttctctcgatacccatcca	7998-8017;	367
	8368	tional		SEQ ID NO. 20	SEQ ID NO. 21	8364-8343
		regulator
lmo0333	936-	Internalin	1778	ccgatttagaaacgcttgga	ttcggcatatcgtgaatcat	1930-1949;	640
	6272			SEQ ID NO. 22	SEQ ID NO. 23	2569-2550
lmo2470	149254-	Internalin	388	tgattccatgcaattactagaacg	aggattctaaactaggtaagtt	149527-149550;	545
	150433			SEQ ID NO. 24	ggtg	150071-150046
					SEQ ID NO. 25
lmo2821	188153-	Internalin	851	tgtaaccccgcttacacagtt	ttacggctggattgtctgtg	188989-189009;	611
	190708			SEQ ID NO. 26	SEQ ID NO. 27	189599-189580

[0047]

TABLE 3
List of bacterial strains examined by PCR using L. monocytogenes virulence specific primers
		lmo0833/			lmo2672/
Strain	Serovar	lmo1188	lmo0834	lmo1116	lmo1134	lmo0333	lmo2470	lmo2821
L. monocytogenes ATCC 19111	1	+	+	+	+	+	+	+
L. monocytogenes ATCC 19112	2	+	+	+	+	+	+	+
L. monocytogenes ATCC 19113	3	+	+	+	+	+	+	+
L. monocytogenes ATCC 19114	4a	−	−	−	−	−	−	−
L. monocytogenes ATCC 19115	4b	−	+	−	+	+	+	+
L. monocytogenes ATCC 19116	4c	−	−	+	−	−	−	+
L. monocytogenes ATCC 19117	4d	−	+	+	+	−	+	+
L. monocytogenes ATCC 19118	4e	−	−	+	+	−	+	+
L. monocytogenes ATCC 15313	1	+	−	+	+	−	+	+
L. monocytogenes EGD (NCTC 7973)	½a	+	+	+	+	+	+	+
L. monocytogenes HCC7	1	+	+	+	+	+	+	+
L. monocytogenes HCC8	1	+	+	+	+	−	+	+
L. monocytogenes HCC12	4	−	−	−	−	−	−	−
L. monocytogenes HCC13	4	−	−	−	−	−	−	−
L. monocytogenes HCC16	4	−	−	−	−	−	−	−
L. monocytogenes HCC17	4	−	−	−	−	−	−	−
L. monocytogenes HCC18	4	−	−	−	−	−	−	−
L. monocytogenes HCC19	4	−	−	−	−	−	−	−
L. monocytogenes HCC23	4	−	−	−	−	−	−	−
L. monocytogenes HCC24	4	−	−	−	−	−	−	−
L. monocytogenes HCC25	4	−	−	−	−	−	−	−
L. monocytogenes 168		+	+	+	+	+	+	+
L. monocytogenes 180		+/−	+	−	+	+	+	+
L. monocytogenes 418		+	+	−	+	+	+	+
L. monocytogenes 742		+	+	+	+	+	+	+
L. monocytogenes 874		−	−	−	−	+	+	+
L. monocytogenes 1002		+	+	+	+	+	+	+
L. monocytogenes 1084		+	+	+	+	−	+	+
L. monocytogenes 1400		+	+	+	+	+	+	+
L. innocua ATCC 33090	6a	−	−	−	−	−	−	−
L. innocua 415		−	−	−	−	−	−	−
L. innocua 416		−	−	−	−	−	−	−
L. innocua 417		−	−	−	−	−	−	−
L. innocua 662		−	−	−	−	−	−	−
L. innocua 1419		−	−	−	−	−	−	−
L. innocua 1425		−	−	−	−	−	−	−
L. innocua 1720		−	−	−	−	−	−	−
L. innocua 1944		−	−	−	−	−	−	−
L. grayi ATCC 19120		−	−	−	−	−	−	−
L. grayi ATCC 25400		−	−	−	−	−	−	−
L. murrayi ATCC 25401		−	−	−	−	−	−	−
L. ivanovii ATCC 19119		−	−	−	−	−	−	−
L. ivanovii 3325		−	−	−	−	−	−	−
L. seeligeri ATCC 35967		−	−	−	−	−	−	−
L. seeligeri 3008		−	−	−	−	−	−	−
L. seeligeri 3321		−	−	−	−	−	−	−
L. welshimeri ATCC 35897		−	−	−	−	−	−	−
L. welshimeri ATCC 43550	½b	−	−	−	−	−	−	−
L. welshimeri ATCC 43551	6a	−	−	−	−	−	−	−
L. welshimeri CCF4		−	−	−	−	−	−	−
L. welshimeri 1471		−	−	−	−	−	−	−
Aeromonas hydrophila ATCC 35654		−	−	−	−	−	−	−
Clostridium perfringens		−	−	−	−	−	−	−
Enterococcus faecalis ATCC 29212		−	−	−	−	−	−	−
Escherichia coli ATCC 25922		−	−	−	−	−	−	−
Flavobacterium indolegenes		−	−	−	−	−	−	−
Klebsiella pneumoniae ATCC 13883		−	−	−	−	−	−	−
Proteus vulgaris ATCC 13315		−	−	−	−	−	−	−
Pseudomonas aeruginosa ATCC 27853		−	−	−	−	−	−	−
Salmonella typhimurium ATCC 14028		−	−	−	−	−	−	−
Serratia marcescens ATCC 8100		−	−	−	−	−	−	−
Staphylococcus aureus ATCC 25923		−	−	−	−	−	−	−
Streptococcus pneumoniae		−	−	−	−	−	−	−
Streptococcus pyogenes ATCC 19615		−	−	−	−	−	−	−
Vibrio cholerae		−	−	−	−	−	−	−
Yersinia pseudotuberculosis		−	−	−	−	−	−	−

[0048] The results indicated that the PCR primers derived from these genes reacted predominantly with virulent strains of L. monocytogenes because the virulence of several of these strains (EGD, 19115, CCF1, HCC7, HCC23 and 15313) was determined previously by mouse virulence assay (Erdenlig et al., 2000). To further verify the virulence of L. monocytogenes strains as determined by PCR, a second mouse virulence trial was recently conducted involving 12 L. monocytogenes strains (Table 4). The validity of PCR determination of the virulence of L. monocytogenes has been again confirmed by the mouse virulence trial. One notable exception is L. monocytogenes strain ATCC153 13, which is avirulent due to a mutation that causes failure to express listeriolysin, a known virulence factor. The PCR results suggest that the other virulence-specific genes in this strain are intact.

TABLE 4
Summary of L. monocytogenes mouse virulence trial
			Mouse
			virulence
Strain	Serovar	PCR	trial	LD50
L. monocytogenes ATCC	2	V	V	1.6 × 109
19112
L. monocytogenes ATCC	4a	A	A	1.9 × 1010
19114
L. monocytogenes ATCC	4b	V	V	6.0 × 108
9115
L. monocytogenes ATCC	4c	V	V	2.6 × 108
19116
L. monocytogenes ATCC	4d	V	V	8.8 × 108
19117
L. monocytogenes ATCC	4e	V	V	7.8 × 109
19118
L. monocytogenes ATCC	1	V	A	>1.2 × 1011
15313
L. monocytogenes EGD	½a	V	V	<1.1 × 107
L. monocytogenes HCC8	1	V	V	<7 × 108
L. monocytogenes	4	A	A	3.5 × 1010
HCC25
L. monocytogenes 874	not	V	V	<8.0 × 107
	determined
L. monocytogenes 1002	not	V	V	5.2 × 108
	determined

[0049] Therefore, the present invention utilizes one or more L. monocytogenes virulence-specific genes that allow detection of virulent strains of L. monocytogenes. Specifically, these genes include lmo0833, lmo2672, lmo1116, and lmo1134 (encoding putative transcriptional regulators); lmo0834, and lmo1188 (encoding proteins with unknown function); and lmo0333, lmo2470, and lmo2821 (encoding proteins similar to internalins). Indeed, the combined use of lmo2470 and lmo1116; or lmo0333 and lmo1116, or the use of lmo2821 alone is sufficient to enable identification of all potentially virulent L. monocytogenes strains under investigation. The scope of this invention also includes other genes identified by the methods described that could indicate virulent forms of L. monocytogenes. For example, the described techniques have been used to identify other genetic markers unique to L. monocytogenes , L. innocua, L. grayi, L. ivanovii, L. seeligeri and L. welshimeri (Table 5), that could be used for the development of species-specific PCR assays. These species-specific PCR assays have been tested against a panel of Listeria and other gram-positive and negative species.

TABLE 5
List of bacterial strains examined in PCR using Listeria species-specific primers
			lmo0733	lin0464	Lgr20-246	Liv22-228	Lse24-315	Lwe7-571
Strain	Serovar	Source	(455 bp)	(749 bp)	(420 bp)	(467 bp)	(371 bp)	(608 bp)
L. monocytogenes ATCC 19111	1	Poultry	+	−	−	−	−	−
L. monocytogenes ATCC 19112	2	Human	+	−	−	−	−	−
L. monocytogenes ATCC 19113	3	Human	+	−	−	−	−	−
L. monocytogenes ATCC 19114	4a	Human	+	−	−	−	−	−
L. monocytogenes ATCC 19115	4b	Human	+	−	−	−	−	−
L. monocytogenes ATCC 19116	4c	Chicken	+	−	−	−	−	−
L. monocytogenes ATCC 19117	4d	Sheep	+	−	−	−	−	−
L. monocytogenes ATCC 19118	4e	Chicken	+	−	−	−	−	−
L. monocytogenes ATCC 15313	1	Rabbit	+	−	−	−	−	−
L. monocytogenes EGD (NCTC7973)	½a	Human	+	−	−	−	−	−
L. monocytogenes HCC7	1	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC8	1	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC12	4	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC13	4	Catfish kidney	+	−	−	−	−	−
L. monocytogenes HCC16	4	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC17	4	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC18	4	Catfish spleen	+	−	−	−	−	−
L. monocytogenes HCC19	4	Catfish spleen	+	−	−	−	−	−
L. monocytogenes HCC23	4	Catfish brain	+	−	−	−	−	−
L. monocytogenes HCC24	4	Catfish spleen	+	−	−	−	−	−
L. monocytogenes HCC25	4	Catfish kidney	+	−	−	−	−	−
L. monocytogenes 168		Aborted calf fetus	+	−	−	−	−	−
L. monocytogenes 180		Human outbreak	+	−	−	−	−	−
L. monocytogenes 418		Freezer study	+	−	−	−	−	−
L. monocytogenes 742		Ground beef	+	−	−	−	−	−
L. monocytogenes 874		Cow brain	+	−	−	−	−	−
L. monocytogenes 1002		Pork sausage	+	−	−	−	−	−
L. monocytogenes 1084		Chicken	+	−	−	−	−	−
L. monocytogenes 1400		Jalisco outbreak	+	−	−	−	−	−
L. innocua ATCC 33090	6a	Cow brain	−	+	−	−	−	−
L. innocua 415		Turkey burger	−	+	−	−	−	−
L. innocua 416		Veal/beef patty	−	+	−	−	−	−
L. innocua 417		Beef steak	−	+	−	−	−	−
L. innocua 662		Raw milk	−	+	−	−	−	−
L. innocua 1419		Ground cheese	−	+	−	−	−	−
L. innocua 1425		Pecorino Romano	−	+	−	−	−	−
L. innocua 1720		Chicken	−	+	−	−	−	−
L. innocua 1944		Ground turkey	−	+	−	−	−	−
L. grayi ATCC 19120		Chinchilla faeces	−	−	+	−	−	−
L. grayi ATCC 25400		Corn leaves/stalks	−	−	+	−	−	−
L. murrayi ATCC 25401		Corn leaves/stalks	−	−	+	−	−	−
L. ivanovii ATCC 19119		Sheep	−	−	−	+	−	−
L. ivanovii 3325		Cheese	−	−	−	+	−	−
L. seeligeri ATCC 35967		Soil	−	−	−	−	+	−
L. seeligeri 3008		Soil	−	−	−	−	+	−
L. seeligeri 3321		Cheese	−	−	−	−	+	−
L. welshimeri ATCC 35897		Plant	−	−	−	−	−	+
L. welshimeri ATCC 43550	½b	Soil	−	−	−	−	−	+
L. welshimeri ATCC 43551	6a	Soil	−	−	−	−	−	+
L. welshimeri CCF4		Catfish brain	−	−	−	−	−	+
L. welshimeri 1471		Environment	−	−	−	−	−	+
Aeromonas hydrophila ATCC 35654			−	−	−	−	−	−
Clostridium perfringens		Clinical	−	−	−	−	−	−
Enterococcus faecalis ATCC 29212			−	−	−	−	−	−
Escherichia coli ATCC 25922			−	−	−	−	−	−
Flavobacterium indolegenes		Clinical	−	−	−	−	−	−
Klebsiella pneumoniae ATCC 13883			−	−	−	−	−	−
Proteus vulgaris ATCC 13315			−	−	−	−	−	−
Pseudomonas aeruginosa ATCC			−	−	−	−	−	−
27853
Salmonella typhimurium ATCC			−	−	−	−	−	−
14028
Serratia marcescens ATCC 8100			−	−	−	−	−	−
Staphylococcus aureus ATCC 25923			−	−	−	−	−	−
Streptococcus pneumoniae		Clinical	−	−	−	−	−	−
Streptococcus pyogenes ATCC			−	−	−	−	−	−
19615
Vibrio cholerae		Clinical	−	−	−	−	−	−
Yersinia pseudotuberculosis		Clinical	−	−	−	−	−	−

[0050] The present invention is also used to detect viable virulent strains of L. monocytogenes. The PCR assay utilized in the present invention is effective in amplifying the above listed gene sequences from chromosomal DNA, which is not effective in distinguishing live L. monocytogenes from dead L. monocytogenes. However, amplification of the above listed gene sequences from mRNA by reverse transcription-PCR (RT-PCR) would only detect the presence of live, viable L. monocytogenes.

[0051] Because transcriptional regulators are essential components in the regulation of RNA synthesis and gene expression within bacteria, and because internalins play vital roles in listerial internalization, they may be potentially useful targets for treatment and control purposes. Therefore, it is also within the scope of this invention to use L. monocytogenes virulence-specific genes (lmo0833, lmo1188, lmo0834, lmo1116, lmo2672, lmo1134, lmo0333, lmo2470, and lmo2821) or their derivatives in the inhibition of growth, reduction of pathogenicity, treatment and prevention of listeriosis caused by virulent strains of Listeria monocytogenes.

[0052] For example, one possible treatment strategy would involve using pharmaceutically active agent(s) that would inactivate or alter the function of one or more of the proteins encoded by the above listed genes, which would either kill the virulent L. monocytogenes or render it susceptible to the host immune system. One possible vaccine strategy would involve altering one or more of the above listed genes or promoter(s) for one or more of the above listed genes such that expression of the encoded protein(s) would be completely disrupted or altered. The said alteration or disruption of expression would render L. monocytogenes avirulent and effective as a live attenuated vaccine.

[0053] While the present invention has been described with reference to specific embodiments and exemplary bacteria species, it will be understood by those skilled in the art that a variety of changes may be made and the substitution of equivalents may be made without departing from the true spirit and scope of the present invention. Many modifications may be made to adapt a particular situation or a particular selected pathogen to the inclusive concept of the present invention. All such modifications or adaptations are intended to be within the scope of the claims appended hereto.

[0054] The complete disclosure of all references cited in this application are fully incorporated herein by reference.

[0055] References

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Claims

1. An isolated virulent gene of L. monocytogenes.

2. The isolated gene of claim 1, wherein said gene encodes a protein having virulent biological activity.

3. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule selected from the group consisting of SEQ ID NOS.: 1-9.

4. The isolated gene of claim 3, wherein said gene encodes a protein having virulent biological activity.

5. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule having 95% sequence homology to a nucleic acid molecule selected from the group consisting of SEQ ID NOS.: 1-9.

6. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule having 90% sequence homology to a nucleic acid molecule selected from the group consisting of SEQ ID NOS.: 1-9.

7. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule having 80% sequence homology to a nucleic acid molecule selected from the group consisting of SEQ ID NOS.: 1-9.

8. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule that binds to a specific primer or probe, said primer or probe being selected from the group consisting of SEQ ID NOS.: 10-27.

9. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule that binds to a polynucleotide fragment having 95% sequence homology to a primer or probe selected from the group consisting of SEQ ID NOS.: 10-27.

10. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule that binds to a polynucleotide fragment having 90% sequence homology to a primer or probe selected from the group consisting of SEQ ID NOS.: 10-27.

11. The isolated gene of claim 1, wherein said gene comprises a nucleic acid molecule that binds to a polynucleotide fragment having 80% sequence homology to a primer or probe selected from the group consisting of SEQ ID NOS.: 10-27.

12. The isolated gene of claim 1, wherein said gene comprises a nucleic acid selected from the group consisting of SEQ ID NOS.: 1-9.

13. A novel primer or probe for the identification of virulent genes of L. monocytogenes, said primer or probe being a polynucleotide fragment of at least 10 base pairs that bind to or are complementary with a portion of at least one polynucleotide selected from the group consisting of SEQ I) NOS.: 1-9.

14. A novel primer or probe for the identification of virulent genes of L. monocytogenes, said primer or probe being selected from the group consisting of SEQ ID NOS.: 10-27.

15. A method of identifying virulent a L. monocytogenes isolate comprising: providing at least one primer or probe specific for a corresponding at least one virulence-specific gene of L. monocytogenes; conducting PCR assay or hybridization using said at least one primer or probe to identify the presence of said corresponding at least one virulence-specific gene in said L. monocytogenes isolate.

16. The method of claim 15, wherein said virulence-specific gene is selected from the group consisting of genes identified by SEQ ID NOS.: 1-9.

17. The method of claim 15, wherein said at least one primer is selected from the group consisting of SEQ ID NOS.: 10-27.

18. The method of claim 15, wherein said at least one primer or probe is two or more primers or probes and said corresponding at least one virulence-specific gene is two or more virulence-specific genes and said PCR assay or hybridization is multiplex polymerase chain reaction or hybridization.

19. The method of claim 15, wherein said PCR assay or hybridization is multiplex polymerase chain reaction or hybridization using said primers or probes specific for said virulence-specific gene in combination with Listeria genus-specific primers or probes or L. moncytogenes species-specific gene sequence.

20. The method of claim 15, wherein said L. monocytogenes species-specific gene sequence is selected from the from the group consisting of genes identified by SEQ ID NOS.: 28-33.

21. The method of claim 15, wherein said PCR assay or hybridization is multiplex polymerase chain reaction or hybridization using said primers or probes specific for said virulence-specific gene in combination with Listeria genus-specific primers or probes and L. moncytogenes species-specific gene sequence.

22. The method of claim 17, wherein said L. monocytogenes species-specific gene sequence is selected from the from the group consisting of genes identified by SEQ ID NOS.: 28-33.

23. The method of claim 15, wherein said at least one virulence-specific gene is involved in inhibition of growth.

24. The method of claim 15, wherein said at least one virulence-specific gene is involved in reduction of pathogenicty.

25. The method of claim 15, wherein said at least one virulence-specific gene is involved in treatment of pathogenicity.

26. The method of claim 15, wherein said at least one virulence-specific gene is involved in the prevention of virulent strains of L. monocytogenes.

27. The method of claim 15, wherein said at least one virulence-specific gene is detected by amplification of said genes from mRNA and said PCR is reverse transcriptase-PCR (RT-PCR).

28. A method of identifying viable virulent strains of L. monocytogenes comprising: providing at least one primer specific for a corresponding at least one virulence-specific gene of L. monocytogenes; using said at least one primer to identify said at least one gene and amplifying sequence of said gene from from mRNA by reverse transcription-PCR (RT-PCR).

29. The method of claim 28, wherein said virulence-specific gene is selected from the group consisting of SEQ ID NOS.: 1-9.

30. A method of treating a host subject in need of treatment for the pathogenic effects of a virulent strain of L. monocytogenes comprising: administering an effective amount of at least one pharmaceutically active agent that is effective in altering or inactivating the function of at least one protein encoded by a virulence-specific gene.

31. The method of claim 30, wherein said virulence-specific gene is selected from the group consisting of SEQ ID NOS.: 1-9.

32. The method of claim 30, wherein said altering or inactivating kills said said virulent strain of L. monocytogenes.

33. The method of claim 30, wherein said altering or inactivating renders said virulent strain of L. monocytogenes susceptible to the immune system of said host subject.

34. A vaccine to protect a subject from the pathogenic effects of a virulent strain of L. monocytogenes comprising: altering said at least one virulence-specific gene so as to render expression of the encoded protein of said at least one gene ineffective, wherein said resulting L. monocytogenes is rendered avirulent and effective as a live attenuated bacteria suitable for use in a vaccine for said virulent strain of L. monocytogenes.

35. The method of claim 34, wherein said virulence-specific gene is selected from the group consisting of SEQ ID NOS.: 1-9.

36. A method of vaccinating a subject to protect the subject from the pathogenic effects of a virulent strain of L. monocytogenes comprising: administering a purified protein encoded by a virulence-specific gene or administering a live viral or bacterial vaccine expressing a protein encoded by a virulence-specific gene or administering a DNA vaccine comprising a virulence-specific gene.

37. The method of claim 36, wherein said virulence-specific gene is selected from the group consisting of SEQ ID NOS.: 1-9.

38. A method of quickly determining if a sample taken from a food product contains a virulent strain of L. monocytogenes, the method comprising: isolating L. monocytogenes from said food sample; providing at least one primer specific for a corresponding at least one virulence-specific gene of L. monocytogenes; conducting PCR assay using said at least one primer to identify the presence of said corresponding at least one virulence-specific gene in said L. monocytogenes isolate.

39. The method of claim 38, wherein said virulence-specific gene is selected from the group consisting of SEQ ID NOS.: 1-9.