Molecular and Cellular Probes (1992) 6, 2 9 1 -297

Identification of invasive Yersinia species using oligonucleotide probes Peter Feng Division of Microbiology, Food and Drug Administration, Washington, DC 20204, USA (Received 14 January 1992, Accepted 11 March 1992)

Oligonucleotide probes directed to the inv and ail invasion genes of Yersinia species were used to analyse yersiniae and non-yersiniae isolates by colony hybridization . The INV-3 probe, targeted to the inv gene of Yersinia pseudotuberculosis, hybridized with all 48 HeLa cell-invasive Y. pseudotuberculosis isolates examined ; the PF-13 probe, specific for the ail gene of Yersinia enterocolitica, identified all invasive strains (36 of 52) of Y . enterocolitica tested . Neither probe hybridized with nonyersinia isolates or other Yersinia species . Southern analyses of restriction enzyme-digested genomic DNA confirmed the specificity of both probes . INV-3 hybridized with a 4. 5 kilobase (kb) Bam HI fragment known to carry the inv gene in Y. pseudotuberculosis . PF-13 was specific for a 1 . 2 kb Cla IAva I fragment in Y. enterocolitica that carried the ail locus . Reactivity with either probe correlated closely with the ability of Y. pseudotuberculosis and Y. enterocolitica isolates to invade HeLa cells .

KEYWORDS: Yersinia spp ., pathogenicity, oligonucleotide probes, invasiveness .

INTRODUCTION to determine the pathogenicity of Yersinia species .', " Most of these tests require the expression of phenotypes encoded by the 70 kb plasmid, which is not stably maintained . The invasion phenotype of yersiniae is generally determined by mammalian tissue culture assays . The ability of Yersinia species to invade tissue cells is chromosomally encoded and therefore stably inherited .', " In this study, oligonucleotide gene probes directed to the invasion gene of Yersinia species were used to examine the homology and distribution of the inv and ail genes among yersiniae and in other invasive enteric pathogens . The probes were also evaluated as an alternative to tissue culture assays for determining the invasive phenotype of Yersinia species .

Pathogenic Yersinia species harbour closely related plasmids of about 70 kilobases (kb) which carry virulence or virulence-associated genes . The plasmid alone, however, does not appear to be sufficient for the expression of full virulence .` Isogenic strains of Yersinia with or without the virulence-associated plasmid showed equal efficiency in invading tissue culture cells .',',' Therefore, chromosomal genes must also contribute to the pathogenicity of Yersinia species . 1 '2s,6 Isberg & Falkow' identified an invasion gene (inv) on the Y. pseudotuberculosis chromosome which encodes for invasin, a 103 kDa protein that binds to cell surface receptors and promotes the entry of yersiniae into eukaryotic cells .' A similar inv locus as well as another gene, ail, which also encodes for the invasion phenotype, have been identified on the Y . enterocolitica chromosome .'- '2 The invasion phenotype encoded by the inv genes from both species enables invasion of several cell lines,'" while that expressed by ail exhibits a more stringent cell line specificity ." , " Several in vitro assays and animal models are used 0890-8508/92/040291 +07 $08 .00/0

MATERIALS AND METHODS Bacterial strains All the bacterial strains used in this study were obtained from the collection maintained by the 291

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Division of Microbiology, Food and Drug Administration, Washington, DC . The species tested are listed in Table 1 .

Oligonucleotide probes The INV-3 probe (5'-GGTCCAGCCTTATTCTGTCTC3') is targeted to a region approximately 180 bases downstream from the 5'- terminus of the Y . pseudotuberculosis inv gene . Oligonucleotide PF-13 (5'GACGCTTCATATGAATAC-3') is directed to a region 60 bases upstream from the 3' end of the ail gene in Y. enterocolitica . Both probe sequences were derived from published data 8'9 and synthesized with an ABI380B DNA synthesizer (Applied Biosystems, Foster City, California) . The oligonucleotide probes were purified by electrophoresis in a 7 M urea-15% polyacrylamide gel in Tris-borate buffer," and desalted with SEP-PAK columns (Millipore, Milford, Massachusetts) ." The probes were 5'-end labelled, using T4 polynucleotide kinase ° and [T- 32 PIATP, and were purified by NACS PREPAC cartridges (GIBCO BRL, Gaithersburg, Maryland) .

DNA hybridization For colony hybridization, bacterial cultures grown overnight on tryptic soy agar plates were replicated onto Whatman No . 541 cellulose filters (Whatman, Maidstone, UK) and lysed by microwave irradiation ." Filters were hybridized overnight at 37 ° C, using 1 x 106 cpm of labelled probe per ml of hybridization

Table 1 . study

solution," and washed twice for 30 min each in 6 x standard saline citrate (SSC)-0-1% sodium dodecyl sulphate (1 x SSC contains 0 , 15m NaCl, 0-015m sodium citrate, pH 7 .0) . The washing temperature used for INV-3 and PF-13 were 59 and 48 ° C, respectively . Hybridized colony blots were visualized by autoradiography, with X-Omat RP X-ray film (Eastman Kodak, Rochester, New York). For Southern analyses, restriction enzymedigested bacterial DNA was separated on a 1 agarose gel and transferred onto nitrocellulose paper (Schleicher & Schuell, Inc ., Keene, New Hampshire) ." Hybridization, washing and autoradiography were performed as described above .

HeLa invasion assay The ability of Yersinia isolates to invade mammalian tissue cells was determined by HeLa cell invasion assays." All tissue culture media and reagents were purchased from GIBCO Laboratories (Life Technologies, Grand Island, New York). HeLa cells maintained on Eagle's minimal essential medium (MEM) with Earle's salts and supplemented with 10% fetal bovine serum (FBS) were seeded into 8-chamber Lab-Tek slides (Nunc, Inc ., Naperville, Illinois) to yield a final cell density of about 2 x 104 cells ml - ' . The slides were held for 24 h at 37°C in a CO 2 incubator to allow monolayer formation . Bacteria resuspended in MEM with Earle's salts + 1 % FBS were seeded to the monolayer to yield a final bacterial concentration of about 1 x 10 8 cfu ml - ' . The monolayer was incubated for 1 h at 37 ° C in a CO2 incubator to allow

Results of probe hybridizations and HeLa cell invasion assays for the bacterial isolates used in this

Reactive of strains

PF-13

HeLa cell invasive

No .

Bacteria

INV-3

Yersinia enterocolitica

52

0

36

36

Yersinia pseudotuberculosis Yersinia frederiksenii Yersinia intermedia Yersinia kristensenii

48 3 4 3

48 0 0 0

0 0 0 0

48 0 0 0

9 7 11 8 2 2

0 0 0 0 0 0

0 0 0 0 0 0

ND* ND ND ND ND ND

Pathogenic Escherichia coli Vibrio spp . Shigella spp .

Salmonella spp . Listeria spp. Aeromonas spp . *ND, not done.

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Identification of invasive Yersinia species infection, and then stained with acridine orange and counterstained with crystal violet ." HeLa cell invasion by Yersinia spp . usually begins with a 0 . 5 h lag period followed by rapid uptake of bacteria for 12 h . 19 The slides were viewed under a fluorescence microscope to determine the presence of intracellular bacteria in the HeLa cell monolayer .

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RESULTS Hybridization studies Colony hybridization analyses showed that both probes were highly specific for the respective Yersinia species . The INV-3 probe (Fig . 1) hybridized with all the isolates of Y. pseudotuberculosis examined, except colony D2 (Fig . 1a) . The identity of colony D2 was retested with API 20E (Analytab Products, Plainview, New York) biochemical tests and determined to be a Y. enterocolitica isolate. Similarly, PF-13 (Fig . 2) hybridized only with Y . enterocolitica and not with other Yersinia species . Of the 52 Y. enterocolitica isolates examined, 36 (69%) were PF-13-positive (Fig . 2b). Neither INV-3 or PF-13 hybridized with other invasive enteric pathogens such as Salmonella, Shigella or enteroinvasive E . coli (Figs 1c and 2c). These results are summarized in Table 1 . To confirm the specificity of INV-3 and PF-13 for the inv and ail genes, respectively, restriction enzyme-digested chromosomal DNA was examined by Southern blot hybridization . INV-3 hybridized only with a 4 . 5 kb Bam HI fragment from Y. pseudotuberculosis (Fig. 3). The specificity of INV-3 for Y . pseudo-

tuberculosis is consistent with the colony hybridization results . In contrast, PF-13 hybridized with a 1 . 2 kb Ava I-Cla I fragment in Y . enterocolitica and a large DNA fragment of about 25 kb in Y. pseudotuberculosis (Fig. 4). No hybridization of PF-13 with Y.

pseudotuberculosis isolates in colony blots was

Fig. 1 . Autoradiogram of colony hybridization of INV-3 probe to yersiniae and non-yersiniae isolates . The colonies on blot (a) are all Y. pseudotuberculosis, except D2 which is Y. enterocolitica . On blot (b), positions A3 to A5 are Y. frederiksenii; A6, A7, B2, and B3 are Y. intermedii; B4 to B6 are Y. kristensenii; all others are Y. enterocolitica. The colonies on blot (c) are non-Yersinia isolates . Colonies A2, 131 in blot (b) and 131, Cl in blot (c) are Y . pseudotuberculosis spotted as positive controls and reference markers . The 4-corner positions of each colony filter are blanks .

observed (Fig . 2a) . DISCUSSION AND CONCLUSIONS HeLa cell invasion assay The results of invasion assays in relation to probe reactivity are summarized in Table 1 . All 48 isolates of Y. pseudotuberculosis that hybridized with the INV-3 probe were invasive for HeLa cells . Similarly, all 36 isolates of Y. enterocolitica that hybridized with PF-13 also invaded HeLa cells . Fluorescence microscopy of HeLa cell monolayers showed numerous intracellular bacteria within 1 h of incubation with the invasive strains (Fig . 5a), whereas no intracellular bacteria were observed when non-invasive yersiniae were used to infect the monolayer (Fig . 5b) .

Colony and Southern hybridization analyses showed both oligonucleotide probes to be highly specific for the respective invasion genes from which they were derived . INV-3, directed to the inv gene of Y. pseudotuberculosis, hybridized only with this species and specifically with a 4. 5 kb Bam HI fragment in the genomic DNA . This result is consistent with previous reports that the 3 . 2 kb inv gene of Y. pseudotuberculosis is located on a 4 . 5 kb Barn HI restriction fragment . 7,8 Interestingly, the INV-3 probe did not hybridize with isolates of Y . enterocolitica, even though the inv genes of Y . pseudotuberculosis and Y. entero-

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Fig. 2. Autoradiogram of colony hybridization of PF-13 probe to yersiniae and non-yersiniae isolates. The colonies on the filters are identical to those described in Fig . 1 .

colitica are 71% identical at the DNA level and the invasin proteins encoded by these genes share similar antigenic epitopes. 10' ",20 The absence of hybridization by INV-3 to Y . enterocolitica may then be explained either by the stringency of the hybridization conditions used or by the sequence of the INV-3 probe, which is apparently specific for a genetic region unique to the my gene of Y. pseudotuberculosis . The PF-13 probe was also very specific, hybridizing only with isolates of Y. enterocolitica and specifically with a 1 . 2 kb Ava I-Cla I fragment known to carry the ail gene in Y. enterocolitica ."' , " Unexpectedly, however, PF-13, which did not hybridize with isolates of Y. pseudotuberculosis on colony blots, did hybridize with a large DNA fragment of about 25 kb in the Southern blot analyses of this species . Although the existence of an ail or ail-like gene in Y . pseudotuberculosis has not been reported, these results are consistent with the findings of Miller et al.," who showed that the 1 . 2 kb Ava I-Cla I fragment carrying

Fig. 3. Autoradiogram of Southern hybridization of INV-3 probe to Bam HI-digested chromosomal DNA . The samples are (A) Y. pseudotuberculosis; (B) Y. enterocolitica; and (C) E . coli. The position and sizes (kb) of lambda DNA digested with Hin dill are marked .

the ail gene from Y. enterocolitica will hybridize with DNA from Y. pestis and Y. pseudotuberculosis . These observations suggest that sequences homologous to the ail gene of Y. enterocolitica are also present in other yersiniae . This evidence, however, does not fully explain why PF-13 hybridizes with Y. pseudotuberculosis DNA on Southern blots but not with the same isolates on colony blots . Perhaps the greater purity and the larger amounts of DNA used in Southern analyses provide more accessible targets for probes than do colony blots, thereby increasing the sensitivity and hybridization efficiency of Southern blots. Neither INV-3 nor PF-13 hybridized with other invasive enteric bacteria or with Y. frederiksenii, Y. intermedii and Y. kristensenii (Table 1), which are generally regarded as non-pathogenic . This suggests that the invasion genes, inv and ail, are present only in the Yersinia spp . that contain the pathogenic



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Fig. 4. Autoradiogram of Southern hybridization of PF-13 probe to Ava I-Cla I-digested chromosomal DNA . The samples are (A) and (B) Y . pseudotuberculosis ; (C) and (D) Y. enterocolitica. The position and sizes (in base pairs) of (pX174 RF DNA digested with Hae III are marked .

losis also appear to carry sequences homologous to ail ."

appears to involve several plasmid and chromosomal encoded genes. One study showed that inv gene sequences are present in clinical isolates of Y . enterocolitica that caused diarrheal disease in humans ." However, others have shown that inv gene sequences are present in both virulent and avirulent isolates ;", " therefore, the presence of inv gene se-

Positive hybridization with INV-3 or PF-13 oligonucleotide probes only shows the presence of inv or ail gene sequences in the probe-reactive Yersinia species . It does not, however, indicate that these species are invasive . The presence of non-functional inv gene sequences in avirulent and non-invasive Y. enterocolitica have been reported ." In this study, however, good correlation was obtained between invasiveness, as determined by HeLa cell assays, and reactivity with either INV-3 or PF-13 probes . These results suggest that these probes may be useful as an alternative to HeLa cell assays for determining the invasive phenotype of Yersinia species . Pathogenicity of Yersinia is highly complex and

quences alone cannot reliably correlate Y. enterocolitica with the occurrence of disease in humans . Several studies, however, have shown that avirulent yersiniae often are not invasive; therefore, the invasion phenotype or the ability of Yersinia species to invade mammalian tissue cells appears to be correlated at least with its pathogenicity in animal models .'-3.' 2.22 To verify this premise, 34 suckling mouse lethal or non-lethal isolates of Y. enterocolitica were tested for hybridization with the PF-13 probe . These bacteria, previously implicated in a foodborne disease outbreak, were isolated from both clinical and food samples and tested by the suckling mouse lethality assay . 24 On colony blots (Fig. 6), PF-13

serotypes . According to other reports, genes homologous to the inv of Y . pseudotuberculosis also exists in Y . enterocolitica and Y. pestis." ," Similarly, although Y. enterocolitica is the only species reported to have the ail gene, Y. pestis and Y. pseudotubercu-

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Fig. 5. HeLa cell invasion assay of yersiniae . HeLa cell monolayer was infected with (a) invasive, and (b) non-invasive Y . enterocolitica .

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Fig. 6. Autoradiogram of colony hybridization of PF-13 probe to suckling mouse-lethal and non-lethal Y . enterocolitica isolates . Three Y. enterocolitica isolates were used as positive controls : A2, strain 8081, serotype 0 :8 ; E7, serotype 0 :3; and F2, strain WA, serotype 0 :8 . A3, E. coli, used as negative control . isolates from colonies 81-88, C1-C8, and D1-D3 were lethal for suckling mice . Isolates from colonies D4-D8, E1-E6, E8, and F3-F5 were non-lethal for suckling mice . All other positions are blanks .

Identification of invasive Yersinia species

hybridized only with the suckling mouse-lethal isolates of Y. enterocolitica . These preliminary findings suggest that these invasion gene-specific oligonucleotide probes can identify invasive yersiniae, but may also be used to determine pathogenicity of Yersinia species .

ACKNOWLEDGEMENTS The author thanks K . A . Lampel and T. A . Cebula for helpful technical discussions and W . E . Hill and L . A. Tomlinson for critical reading of the manuscript . Also, special thanks are given to V . L. Miller for providing the ail gene sequence .

REFERENCES 1 . Heesemann, J ., Algermissen, B . & Laufs, R . (1984) . Genetically manipulated virulence of Yersinia enterocolitica . Infection and Immunity 46, 105-10 . 2 . Portnoy, D . A ., Moseley, S . L . & Falkow, S . (1981) . Characterization of plasmid and plasmid-associated determinants of Yersinia enterocolitica pathogenesis . Infection and Immunity 31, 775-82 . 3. Schiemann, D . A . (1989) . Yersinia enterocolitica and Yersinia pseudotuberculosis. In Foodborne Bacterial Pathogens (Doyle, M ., ed .), pp . 601-72 . New York and Basel : Marcel Dekker Inc . 4 Rosqvist, R. & Wolf-Watz, H . (1986) . Virulence plasmidassociated HeLa cell induced cytotoxicity of Yersinia pseudotuberculosis . Microbial Pathogenesis 1, 229-40. 5. Schiemann, D . A . & Devenish, J . A . (1982) . Relationship of HeLa cell infectivity to biochemical, serological, and virulence characteristics of Yersinia enterocolitica . Infection and Immunity 35, 497-506 . 6. Une, T . (1977) . Studies on the pathogenicity of Yersinia enterocolitica . II . Interaction with cultured cells in vitro . Microbiology and Immunology 21, 365-77 . 7. Isberg, R . R . & Falkow, 5 . (1985) . A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli k-12 . Nature 317, 262-4 . 8. Isberg, R . R ., Voorhis, D . L . & Falkow, S . (1987) . Identification of invasin : a protein that allows enteric bacteria to penetrate cultured mammalian cells . Cell 50, 76978. 9. Miller, V . L ., Bliska, J . B . & Falkow, S . (1990). Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the ail protein product . Journal of Bacteriology 172, 1062-9. 10. Miller, V . L . & Falkow, S . (1988) . Evidence for two loci in Yersinia enterocolitica that can promote invasion of epithelial cells . Infection and Immunity 56, 1242-8 .

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11 . Miller, V . L., Farmer III, J . J ., Hill, W . E. & Falkow, S . (1989) . The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease . Infection and immunity 57, 121-31 . 12 . Miller, V . L ., Finlay, B . B . & Falkow, S . (1988) . Factors essential for the penetration of mammalian cellls by Yersinia . Current Topics in Microbiology and Immunology 138, 15-39 . 13 . Maniatis, T ., Fritsch, E. F . & Sambrook, J . (1982). Molecular Cloning : A Laboratory Manual. Cold Spring Harbor, New York : Cold Spring Harbor Laboratory . 14. Sanchez-Pescador, R . & Urdea, M . S . (1984) . Laboratory methods : use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing. DNA 3, 339-43 . 15 . Datta, A . R ., Wentz, B . A . & Hill, W . E . (1987). Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization . Applied and Environmental Microbiology 53, 2256-9 . 16 . Hill, W . E., Wentz, B . A., Payne, W. L ., Jagow, J . A . & Zon, G . (1986) . DNA colony hybridization method using synthetic oligonucleotides to detect enterotoxigenic Escherichia coli : collaborative study . Journal of Association of Official Analytical Chemists 69, 531-6. 17. Southern, E . M . (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis . Journal of Molecular Biology 98, 503-17 . 18. Miliotis, M . D . (1991) . Acridine orange stain for determining intracellular enteropathogens in HeLa cells . Journal of Clinical Microbiology 29, 830-1 . 19 . Bovallius, A . & Nilsson, G . (1975). Ingestion and survival of Yersinia pseudotuberculosis in HeLa cells . Canadian Journal of Microbiology 21, 1997-2007 . 20. Pepe, J . C . & Miller, V . L . (1990) . The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin . journal of Bacteriology 172, 3780-9 . 21 . Robins-Browne, R ., Miliotis, M. D ., Cianciosi, S ., Miller, V . L., Falkow, S . & Morris, J . G . (1989). Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species . Journal of Clinical Microbiology 27, 644-50 . 22 . Pierson, D . E . & Falkow, S . (1990) . Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv-homologous sequences . Infection and Immunity 58,1059-64. 23 . Morris, J . G ., Prado, V ., Ferreccio, C . et al . (1991) . Yersinia enterocolitica isolated from two cohorts of young children in Santiago, Chile : incidence of and lack of correlation between illness and proposed virulence factors . Journal of Clinical Microbiology 29, 2784-8 . 24. Aulisio, C . C. G ., Stanfield, J . T ., Weagent, S . D . & Hill, W. E . (1983). Yersinosis associated with tofu consumption : serological, biochemical and pathogenicity studies of Yersinia enterocolitica isolates . Journal of Food Protection 46, 226-30.

Identification of invasive Yersinia species using oligonucleotide probes.

Oligonucleotide probes directed to the inv and ail invasion genes of Yersinia species were used to analyse yersiniae and non-yersiniae isolates by col...
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