Role of Yersinia Enterocolitica Yst Toxin in Experimental Infection of Young Rabbits ISABELLE DELOR and GUY R

Home , Yersiniosis

INFECMION AND IMMUNITY, OCt. 1992, p. 4269-4277 Vol. 60, No. 10 0019-9567/92/104269-09$02.00/0 Copyright C 1992, American Society for Microbiology Role of Yersinia enterocolitica Yst Toxin in Experimental Infection of Young Rabbits ISABELLE DELOR AND GUY R. CORNELIS* Microbial Pathogenesis Unit, Intemnational Institute of Cellular and Molecular Pathology and Faculte de Medecine, Universit6 Catholique de Louvain, UCL 54.90, B-1200 Brussels, Belgium Received 14 May 1992/Accepted 31 July 1992

We constructed a Yst-negative mutant of Yersinia enterocolitica W1024 by reverse genetics, and we compared the virulence of the yst+ and yst isogenic strains in an experimental oral infection of the young rabbit. The rabbits infected with theyst+ strain suffered from diarrhea and lost weight, and most ofthem died. By contrast, the occurrence of diarrhea, weight loss, and death in the group of rabbits infected with the yst mutant was as low as that in the group ofuninfected rabbits. Bacteria from both strains were excreted in the feces and induced a serum antibody response against Yop proteins. Theyst mutant disappeared more rapidly from the feces. We conclude that the enterotoxin Yst is a major factor involved in the Y. enterocolitica-associated diarrhea in the young rabbit. Given the similarity with the symptoms observed for children, this result suggests that Yst could also be an important factor in diarrhea in young children infected with Y. enterocolitica.

Yersinia enterocolitica is an enterobacterium frequently nization in three domains (pre-, pro-, and mature toxin) involved in human self-limiting enterocolitis. At least in resembles that of the enterotoxin STI (14, 33, 44), but Europe, it is a common pork contaminant (61), and the although the precursors have the same size, the mature Yst disease often results from consumption of raw or under- toxin is much larger than STI. The degree of conservation cooked pork in the 2 weeks preceding illness (58). The between the two toxins is greater in the mature proteins than predominant clinical features in children under 5 years of age in the other domains. In particular, the active site, formed of are diarrhea, fever, and, less frequently, abdominal pain. A 13 amino acids including six cysteines, is highly conserved in mesenteric adenitis characterized by fever, abdominal pain, Yst and STI as well as in all related heat-stable toxins (57, and leukocytosis is common in older children and adoles- 63). cents (21, 27, 59, 60; for a review, see reference 7). Interestingly, only the Y enterocolitica strains that belong Bacterial resistance to the nonspecific immune response is to the pathogenic serotypes possess the yst gene (11) and governed by a 70-kb plasmid called pYV, which is also most, if not all, fresh human isolates express Yst (39). By present in Y pseudotuberculosis and Y pestis, two related contrast, many collection strains no longer produce entero- pathogens having a strong tropism for lymphoid tissue (2, 42, toxin-(3, 4, 39), but these strains, nevertheless, carry an 64). The pYV plasmid and the antihost Yop proteins that it intact but silent yst gene (9, lOa). encodes are thus presumably responsible for the mesenteric The similarity between Yst and STI suggests that Yst adenitis often observed with Y enterocolitica (for a review, could be responsible for the diarrhea induced by Y entero- see reference 8). colitica. At variance with Y. pseudotuberculosis and Y pestis, Y However, the following observations argue against enterocolitica secretes a chromosome-encoded heat-stable a role for this enterotoxin in the pathogenesis of diarrhea: (i) enterotoxin called Yst (37). This enterotoxin, detectable in attempts to demonstrate enterotoxin in the watery intestinal broth culture supernatants by the infant mouse test, consists contents of diarrheic animals infected by Y enterocolitica of a 30-amino-acid peptide (56). Its physicochemical and have been unsuccessful (38, 46, 48); (ii) Yst can only be antigenic properties as well as its mode of action are similar detected in supernatants of late-log-phase cultures of Y to those of the heat-stable toxin STI of Escherichia coli (3, enterocolitica incubated at temperatures lower than 30°C (3, 13, 32, 37). Both toxins activate the particulate form of 45). The role of Yst thus remains a matter of debate. intestinal guanylate cyclase and induce a fluid accumulation The mouse is often used to study the pathogenesis of by increasing the concentration of cyclic GMP in intestinal yersiniae. After oral inoculation, Y enterocolitica invades epithelial cells (43, 47). The Y enterocolitica Yst enterotoxin the Peyer's patches, the mesenteric lymph nodes, and, also increases intracellular cyclic GMP levels in cultured depending on the experimental conditions, the spleen and cells lines (17, 31, 45). the liver (4). However, although diarrhea has been described According to the nucleic acid sequence, Yst is synthesized for Swiss albino mice infected with Y enterocolitica (22, 49, as a 71-amino-acid polypeptide (11). The C-terminal 30 50), it is not a major symptom in the mouse model. By amino acids correspond to the toxin extracted from culture contrast, the infection of the young rabbit resembles that of supematants (11, 56). The N-terminal 18 residues have the child: the oral inoculation induces both a clear diarrhea properties of a signal sequence. The central 22 residues are and a systemic invasion (16, 24, 34, 35, 38). presumably removed after the secretion process. This orga- In this paper, we report the engineering of an enterotoxin- negative Y enterocolitica mutant by reverse genetics, and we present a comparative study of this yst mutant and the * Corresponding author. Electronic mail address: Cornelis@ wild-type strain in an experimental infection of young rab- mipa.ucl.ac.be. bits. 4269 4270 DELOR AND CORNELIS INFECr. IMMUN.

TABLE 1. Plasmids used in this study Plasmid Genotypic or relevant characteristics Source or reference pBC19R pTZ19R onTRK2 China et al. (5) pHV100 E. coli Plac, luxAB bla+ Peabody et al. (41) pIC20H bla', multiple cloning site Marsh et al. (29) pID6 pBC19R + 5-kb EcoRI-PstI fragment containing yst from Y enterocolitica W1024 Delor et al. (11) pID10 212-bp DraI-HindIII fragment ofyst inserted in HindII-HindIII sites of pBC19R; yst probe Delor et al. (11) pID29 pPolyIII-D + 5-kb EcoRI-PstI fragment of pID6 This paper pID30 pIC20H + 2.4-kb HindIII cassette containing promoterless luxAB genes This paper pID32 pID29 + promoterless lux4B cassette cloned in HindIII site located within yst This paper pID101 pKNG101 + 7.5-kb NotI fragment of pID32 containing the yst'-lux4B-yst' disrupted gene This paper pKNG101 pir-ofR6K oriTRK2 strAB sacBR; replicates only in pir+ host strain Kaniga et al. (18) pPolyIII-D bla oripBR322; polylinker flanked by SfiI and NotI Lathe et al. (23)

MATERIALS AND METHODS plasmid was then inserted in the HindIII site localized within Bacterial strains and plasmids. Y enterocolitica W1024 yst of pID29 to obtain pID32. Plasmid pID32 thus contains (serotype 0:9) (kind gift of G. Wauters, Brussels, Belgium) lux4B coding for an active luciferase and transcribed from was isolated in 1988 from human stools. For selection the yst promoter. purposes, this strain was made nalidixic acid resistant. In a second step (Fig. 1B), we transferred the mutated yst KNG1024 is a derivative of strain W1024 in which the blaA gene on the suicide vector pKNG101, which contains the gene encoding 3-lactamase A (6, 51) has been replaced by sacB gene encoding levansucrase and conferring sensitivity the luxAB gene marker (18). ID1024 is an enterotoxin- to sucrose (18). The NotI fragment from pID32 containing negative mutant of W1024 constructed in this study. E. coli the yst'-lux4B-yst" construct was introduced in the unique SmlO lambda pir+, constructed by Miller and Mekalanos NotI site of pKNG101 to give pID101. (30), allows the replication of pir mutants of R6K and Finally, the chromosomal yst gene of strain W1024 was mobilizes plasmids containing the origin of transfer of RK2. replaced by the disrupted yst'-luxAB-yst" gene (Fig. 1C). Plasmids are listed in Table 1. Plasmid pID101 was mobilized in Y enterocolitica W1024. Media and growth conditions. Bacteria were routinely Its integration in the Y enterocolitica chromosome, result- grown in tryptic soy broth (TSB) (GIBCO Diagnostics, ing from a single homologous recombination, was obtained Madison, Wis.) and plated at 28°C on tryptic soy agar (TSA) by selecting for the streptomycin resistance marker of the (Diagnostics Pasteur, Mames la Coquette, France) enriched plasmid. The integration was checked by Southern hybrid- with 0.3% yeast extract (Difco Laboratories, Detroit, ization with yst and luxAB probes (Fig. 2), and the relevant Mich.). Selective agents were nalidixic acid (35 ,ug/ml), clone was then cultured without selection pressure to allow ampicillin (150 ,ug/ml), streptomycin (50 ,ug/ml), and sucrose the second recombination to occur. The second recombina- (5%). tional event was selected by plating on TSA supplemented For enterotoxin production, bacteria were grown for 48 h with 5% sucrose and deprived of streptomycin. Surviving at 28°C with vigorous shaking in 10 ml of TSB contained in colonies were tested by replica plating on streptomycin. a 100-ml conical flask. The culture was centrifuged for 10 Genetic exchange, evidenced by streptomycin sensitivity min at 8,000 x g, and the supernatant was assayed, either and luciferase production, occurred in 16% of the colonies directly or after storage at -20°C, in newborn mice. surviving on sucrose. DNA from a few of these colonies was To monitor the production of the Yop proteins (Yops), analyzed by Southern hybridization (Fig. 2). bacteria were grown in BHI-Ox, which consists of brain Characterization of the Y. enterocolitica strains. The Ca2+ heart infusion broth (Difco) supplemented with 20 mM dependency for growth at 37°C and the capacity to produce sodium oxalate, 20 mM MgCl2, and 4% (wt/vol) glucose. Yops and YadA were checked as described by Comelis et al. Engineering of the Yst-negative mutant ID1024. As a first (9). Briefly, 10-ml cultures in BHI-Ox were incubated for 2 h step (Fig. 1A), we inserted a luxAB cassette in the HindIII at room temperature and then for 4 h at 37°C. The superna- site localized at the beginning of the sequence encoding the tant of the cultures was then concentrated by (NH4)2SO4 mature Yst toxin. A 5-kb EcoRI-PstI fragment from pID6 precipitation and analyzed by sodium dodecyl sulfate-poly- containing theyst gene and surrounding chromosomal DNA acrylamide gel electrophoresis (SDS-PAGE). from Y enterocolitica W1024 (11) was inserted in the EcoRI The enterotoxin was assayed in suckling mice as described and PstI sites of pPolyIII-D in order to gain NotI sites (23). by Dean et al. (10). Groups of three 2- to 5-day-old suckling The resulting plasmid was called pID29. A PvuIl-SalI frag- mice were injected intragastrically through the abdomen ment of pHV100 (41) containing luxAB was cloned in the with 0.1 ml of test substance supplemented with 0.1% Evans Sall and EcoRV sites of pIC20H (29) in order to gain HindIll blue dye as a marker. Mice were kept for 3 h and then sites. The luxAB HindIII cassette of the resulting pID30 sacrificed by inhalation of chloroform. The intestines were VOL. 60, 1992 ROLE OF Y ENTEROCOLITICA Yst TOXIN 4271 Si ~~E

Placri ~~~~~~~~~~~~~~~~~~LuxA Plac~~~~~~~~~~~~~~~~~~~~~~lc LuxB5 ptD6 ApPv pHV100 Hc' gkb ~~~~~~~~~~~~~~~~~~~~~~5.2kb AkbApREPvP Pv 2.6kb E

H Hc N N

ApR 7kb~~~~APRacOuxE H

2kb~ ~~~~~~7k k PyD HccplD32 H

\Oni MOS N p= Q > S rsacBR \ >Plac

NE H MC N N S v

Hc mobSB BRpID32 c K2 Sat\ 9.k- L pKNGIO M Hc luxB/ H ri 6.8kb MCS Hc R Sm

st.~rB SAyH Hc E~u A~ N ~~B .H.---- wild-type

mR SacBR~saBR

obstrAN\4 ~~~~~~~~~~~luxAB K2~~~ ~ ~ ~ ~ SucR, Lukt Si ~~()SmR, str ori (b)~(b -wid-yp

FIG. 1. Engineering of theyst mutant ID1024: (A, B, and C) the three steps outlined in Materials and Methods. Only the restriction sites used for the cloning are indicated. Hatched boxes represent Y. enterocolitica W1024 chromosome. The yst gene is represented by black boxes. PIac indicates the E. coli lac promoter. The sizes of plasmids are indicated in kilobases. on, replication origin from pBR322; MCS, multiple cloning site; Ap, ampicillin; Sm, streptomycin; Suc, sucrose; luL4AB, genes from Vibno harveyi encoding luciferase; E, EcoRI; Ev, EcoRV; H, HindIII; N, NotI; P, PstI; Pv, PvuII; S1, Sail. 4272 DELOR AND CORNELIS INFECT. IMMUN.

A B The day before inoculation, we compared the rabbits to each other with respect to weight and weight change. We 1 2 3 1 23 then formed 19 pairs of animals presenting the same profile, and one member of each pair was assigned to group B (mean weight ± standard deviation, 825 ± 103 g), while the other was assigned to group C (mean weight, 807 ± 109 g). The last eight rabbits weighing 919 ± 116 g constituted group A. Cages of group A and of group B rabbits were separated but placed in the same room. Group C was housed in a separate room. The quarantine was pursued for 1 day during which one rabbit of group C died without any clear symptom of diarrhea or infection. From the beginning of the experiment, separate material, clothes, and shoes were used for handling each group. Waste products were decontaminated daily. - 2.6kb Experimental design of the infection. Aliquots (50 ,ul) of a fresh culture of Y enterocolitica KNG1024 or ID1024, grown in TSB at room temperature, were plated on TSA. 1.7kb 1.7kb --- After 20 h of incubation at 28°C, bacteria were scraped from 1.4kb - the plates and washed in phosphate-buffered saline (PBS; 50 mM sodium phosphate, 150 mM NaCl [pH 7.4]). The turbid- ity of the suspension was adjusted to an optical density at 600 nm of 1.0, corresponding to 1.6 x 109 CFU ml-'. The bacterial viable count was checked by plate enumeration. Rabbits were intubated with a 40-cm-long XRO feeding tube (Vygon, Ecouen, France) connected to a syringe. They It eqt ll RT 44 et C%IC4 04 C4 N040 C14 were inoculated intragastrically with 10 ml of bacterial suspension containing 1.6 x 1010 CFU or with PBS just before receiving their daily diet. Each rabbit was weighed once every 2 or 3 days. Evidence i5 of diarrhea was assessed daily. Rabbits were considered to FIG. 2. Southern hybridization analysis of the engineered have diarrhea when the feces were semisolid and the perinea strains. Chromosomal DNA was digested by HindII. (A) luxB or hind legs were wet and soiled. Feces from each rabbit probe; (B) yst probe. Lanes: 1, strain W1024 after integration of yst'-luxAB-yst' (diploid strain); 2, W1024 wild-type; 3, strain ID1024 were collected every other day by gentle pressure on the resulting from the excision of the yst wild-type allele. The W1024 rectum. In case of diarrhea, the feces were sampled on a wild-type DNA hybridized only with the yst probe at the level of a swab. Blood samples were drawn at day 5 before inoculation 2.6-kb fragmnent, and the ID1024 DNA hybridized with theyst probe and at days 16 and 25 after inoculation. Surviving animals at the level of a 1.7-kb fragment and with the luxAB probe at the were sacrificed at day 25 by injection of an overdose (70 levels of 1.7- and 1.4-kb fragments. For the diploid 1024 strain, all mg/kg of body weight) of sodium pentobarbital (Nembutal; the bands described above are present. Sanofi, Brussels, Belgium) in the marginal vein of the ear. From day 8 after inoculation, spleens from dead or sacrificed rabbits were removed for bacterial cultures. removed and weighed, and the remaining bodies were The food consumption in each cage was monitored daily weighed. The ratio of the weight of the intestine to the by weighing the given and eventually remaining diet. weight of remaining body gave a measure of the fluid Spleen cultures. Spleens were homogenized in 10 ml of accumulation. Values greater than 0.083 were considered sterile saline by a 30-s treatment with a mixer (model positive. Ultra-Turrax TP18/10; Janke and Kunkel Ika, Staufen, Ger- DNA manipulations. Plasmid DNA was prepared as de- many). Spleen suspensions were centrifuged at 2,000 x g for scribed by Balligand et al. (1). Chromosomal DNA was 10 min. Pellets were resuspended in saline solution, plated at prepared essentially as described by Marmur (28). Southern appropriate dilutions on medium supplemented with nali- hybridization analysis was done according to standard meth- dixic acid, and incubated at 28°C. ods (25, 55). The yst probe was a 220-nucleotide BamHI- Bacterial numeration in feces. Feces (100 to 300 mg) were HindIII fragment from pID10, and the luxAB probe was a resuspended in 5 ml of TSB, mixed with a vortex mixer until 2-kb PvuII-SalI fragment from pHV100. Probes were puri- the suspension was homogenized, and allowed to sediment fied and labeled as described previously (12). on ice. Bacteria were counted in the upper phase. The Animals, housing, and diet. Fifty male and female weanling luciferase activity of strain KNG1024 was high enough to New Zealand White rabbits were used. Groups of three or allow the quantification of the bacteria by luminometry (19). four animals were housed in the same cage (45 by 46 by 32 For the luminometric assay, a 250-,l volume of the upper cm). They were first observed and acclimated to laboratory phase was mixed with 50 ,ul of a suspension of 0.1% conditions for a period of 9 days. Four rabbits died during n-decanal (Sigma, St. Louis, Mo.) in water, and the bacteria this quarantine. Each cage was identified by a letter, and were counted in a Pico-lite luminometer (Packard Instru- each rabbit was identified by a number. The temperature was ment Co., Downers Grove, Ill.), as described by Kaniga et 21°C. Drinking water was available ad libitum. The diet, al. (19). The values in counts per minute milliliter-' were distributed every morning, consisted of complete rabbit food converted to CFU also as described previously (19). (AII, 52; Dossche, Leuven, Belgium). The initial daily dose The bacteria were also counted by plating appropriate of 30 g rabbit-1 was increased by 5 g every day. dilutions (based on the luminometric assay when possible) VOL. 60, 1992 ROLE OF Y ENTEROCOLITICA Yst TOXIN 4273 on TSA supplemented with nalidixic acid and incubating at and C). Most rabbits from group C began to excrete 280C. KNG1024 bacteria 48 h after inoculation (Table 2); the Immunoblot analysis. The presence of anti-Yop antibodies number of bacteria in the excretion reached a maximum in rabbit sera was monitored by Western blotting (immuno- around day 6 or 7, and the presence of bacteria persisted blotting) as described by Sory et al. (54) for mice sera, until the day of death or sacrifice. Four of the 18 rabbits except that the bands were revealed by incubation with never excreted Y enterocolitica. The number of ID1024 anti-rabbit immunoglobulin-peroxidase conjugate (Dako- bacteria excreted by group B was quantitatively less impor- patts) diluted 1/200 in blocking buffer. tant and decreased more rapidly (Table 2). Seven of the 19 rabbits never excreted Y enterocolitica or excreted only RESULTS very few bacteria. However, there was a high variability in bacterial excretion for both groups B and C. We never Construction of a pair of Yst producer and nonproducer Y. isolated Y enterocolitica from feces samples from group A enterocolitica strains. We constructed a Y enterocolitica (Table 2). strain that does not produce the heat-stable toxin Yst, by The majority of rabbits from group C lost weight during reverse genetics as detailed in Materials and Methods and the infection. For 11 rabbits, the weight loss was associated schematized in Fig. 1. Briefly, the wild-typeyst gene from Y with diarrhea. For six rabbits, the weight loss preceded enterocolitica W1024, previously cloned on a pTZ19 deriv- diarrhea, and among these rabbits, three never excreted Y ative (11), was inactivated by insertion of a luxAB cassette. enterocolitica. The last rabbit of this group suffered from The mutated yst allele was then subcloned in plasmid diarrhea without weight loss. Globally, the animals in group pKNG101, a suicide vector containing the sacB gene, in B gained weight during the infection. Only four rabbits lost order to facilitate the selection of the allelic exchange (18) weight during a few days, but these rabbits never suffered and finally reintroduced into Y enterocolitica W1024. The from diarrhea and only two of them excreted Y enteroco- two successive homologous recombinations leading to the litica. All rabbits in group A gained weight, except the one allele replacement ofyst byyst'-luxAB-yst" were monitored that suffered from diarrhea. A graphic of cumulative weight by Southern hybridization analysis with luxAB and a frag- loss during the experiment is presented in Fig. 3B. ment of yst as probes (Fig. 2). The engineered yst mutant, Fourteen rabbits in group C died, while only two rabbits in called ID1024, expressed luxAB from the yst promoter. group B died and none of the rabbits in control group A died. As a Yst producer strain, we used KNG1024, another There was a high variability in the day of death. Figure 3C derivative of strain W1024 in which the blaA gene, encoding presents a comparative graphic of the cumulative mortality ,-lactamase A, has been replaced by the lux4B marker in the three groups. expressed from the Pwc promoter (18). Because of the The animals that died after day 8 were dissected, and the difference in the strength of the promoters serving luxAB, spleens were removed and cultured. Six spleen cultures from strains KNG1024 and ID1024 could easily be distinguished group C were positive. The CFU ranged from 5.5 x 102 to 6 by luminometry. The strong luciferase activity of KNG1024 x 10 per spleen. For group B, the spleen culture of the only allows the quantification of the bacteria in feces samples rabbit that died (day 14) was negative. All the spleens from (19). It is not the case for ID1024. rabbits that were sacrificed on day 25 were sterile in both Prior to use, we analyzed KNG1024 and ID1024 for the groups. presence of the classical Y enterocolitica virulence markers: The presence of anti-Yop antibodies was monitored by Ca2' requirement for growth at 37°C, secretion of Yop Western blotting with sera sampled 5 days before bacterial proteins, and production of enterotoxin. The responses inoculation and 16 and 25 days after infection (Table 2). given by these two strains were identical, except in the infant None of the preinfection sera contained anti-Yop antibodies. mouse test, which was positive (ratio of 0.105) for KNG1024 For group C, all the sera from the surviving animals (eight and negative (ratio of 0.062) for ID1024. serum samples at day 16 and four serum samples at day 25) Experimental infection of rabbits. A group of 18 rabbits were positive. For group B, 12 of 17 serum samples reacted (group C) were inoculated intragastrically with 1.6 x 1010 against Yop proteins. Four serum samples tested from group CFU of KNG1024 (Yst producer strain). A group of 19 A remained negative. rabbits (group B) were inoculated with 1.5 x 1010 CFU of Control of the bacterial strains. Colonies of Y enteroco- ID1024 (Yst-negative strain). A control group of eight rabbits litica KNG1024 and ID1024 Were recovered regularly from (group A) received 10 ml of sterile PBS solution. The the feces and, after the death of the animal, from the spleen. duration of the experiment was 25 days. The details of the The colonies growing on nalidixic acid plates were identified daily course of clinical symptoms in the rabbits are pre- on the basis of their luciferase production. The colonies of sented in Table 2. KNG1024, expressing luxAB from the PIac promoter, emit- In group C, enteritis was the major clinical symptom: by ted enough light to be seen in a dark room. The colonies of 21 days after inoculation, all the rabbits of this group had strain ID1024 could only be identified by autoradiography. suffered from diarrhea. The gastroenteritis generally lasted At the end of the experiment, several clones in samples for 7 days, was associated with Y enterocolitica excretion collected from rabbits at different stages of infection were (14 of 18 rabbits), and often ended with the death of the analyzed. All the clones analyzed required Ca2" for growth rabbit (10 of 14 rabbits). By contrast, only 4 of 19 rabbits in at 37°C and synthesized Yop proteins. The Southern hybrid- group B developed diarrhea; two of these died with diarrhea, ization profile of chromosomal DNA probed withyst as well but the diarrheic stools did not contain detectable Y entero- as the responses in the infant mouse test correlated with the colitica (Table 2). Only one 1 of 8 rabbits in group A suffered strains used for inoculation. Since the two strains could be from diarrhea (Table 2). A graphic of cumulative occurrence distinguished on the basis of their specific luminometric of diarrhea during the experiment is presented in Fig. 3A. activity, we also checked the identity of the strains by To monitor the bacterial colonization of the intestine, we monitoring the luciferase activity with a luminometer. This analyzed the feces of every rabbit approximately every other activity was in perfect agreement with the identity of the day, by luminometry (group C) and viable counts (groups B strain used to inoculate the rabbit (data not shown). Hence, 4274 DELOR AND CORNELIS INFECT. IMMUN.

TABLE 2. Course of clinical symptoms in rabbitsa Bacterial counts in the feces (log 10 CFU g-1) and incidence of diarrhoea (shaded area) Anti-Yop Ab DaysO0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 16 25 Group Rabbit A AO _II S Al _|2 S + A2 _N| S A3EO ND NIN S

co .S-

B DO ND ND ND ND ND ND ND ND 5.0 6.8 6.4 S + + DI 2.5 2.8 ND 3.0 3.6 44 24 2.8 ND ND S + + D2 ND ND ND ND ND ND ND ND ND ND S- EO ND ND ND ND 2.7 ND 1.3 ND ND ND ND S + + El ND ND ND ND ND ND ND ND ND ND ND S - E2 ND ND ND ND ND 3.1 3.8 ND S + + E3 FO 4.2 5.1 6.2 7.4 7.8 8.0 7.3 7.5 6.3 4.6 S + + Fl 4.2 3.8 5.0 6.3 7.2 8.4 7.9 7.7 5.5 4.5 ND S + + F2 3.2 4.9 5.5 6.4 6.6 6.4 7.2 7.5 3.9 3.6 S + + F3 ND 3.0 ND ND ND 2.8 2.5 3.2 ND 5.0 4.1 3.5 S + + GO ND ND ND ND ND ND ND ND ND ND ND ND S _ Gl 2.8 3.8 5.0 4.9 5.3 4.4 4.4 3.6 ND ND ND S + + G2 ND ND ND ND ND ND ND ND 1.2 4.2 S G3 ND ND 2.5 2.4 ND ND ND ND ND ND S

HI 4.5 2.5 3.8 5.1 5.8 5.1 4.8 3.2 ND + H2 2.5 3.8 3.6 4.5 5.1 3.8 2.9 ND ND 1 + H3 ND 3.6 4.3 5.8 5.9 6 5.3 4.4 3.9 ND ND _ s _ 10 3_ 5.2 p 5.7 6.3 6.8 = =+~___ -

< 12 _ 4.71 = Il

6. 3.

|L2 _

||M3 ND ND_ ND ND2.7 3-ND- 50|63|,ND ND- ND ND _ ND ND + __zI.7KM2MOJ2l3 N2.3 T33 N4 N5 ND6 N L+ £zL__+ _..z__z._r__._J~.X._ __S +

IND, not detected; D, death of the animal; S, sacriticed. Groups: A, uninfected; B, infected with yst mutant; C, infected withyst' strain.

the strains isolated from the rabbits corresponded to the susceptibility of rabbits to Y enterocolitica appeared, how- strains used for inoculation, and no cross contamination ever, to be more variable in this study than in others occurred during the experiment. describing experimental yersiniosis. We observed an important individual variation for the onset of diarrhea and for DISCUSSION bacterial excretion. This could be the consequence of our use of rabbits slightly older than those used in previous In this comparative experiment using a pair ofyst+ andyst studies. Y enterocolitica is indeed known to induce diar- isogenic Y enterocolitica strains, we confirmed that diarrhea rhea, particularly in very young animals (38, 46). In addition, is the major clinical symptom presented by the young rabbit since our rabbits were housed in groups of four, we cannot infected with yst+ Y enterocolitica. The general clinical exclude that a secondary infection occurred, from an animal evolution of our young rabbits orally infected with the highly infected to an animal having resisted the experimental wild-type strain was similar to that reported in previous inoculation. The 14 cases of diarrhea were associated with studies (16, 34, 38): all the rabbits suffered, at some stage, weight loss, and 10 ended with the rabbits' deaths. Pai et al. from diarrhea, and diarrhea in 14 of 18 rabbits was associ- (38) observed a similar result. These observations confirm ated with Y enterocolitica excretion and weight loss. The that diarrhea is particularly exhausting for the young rabbit VOL. 60, 1992 ROLE OF Y ENTEROCOLITICA Yst TOXIN 4275 A and it induced a serum antibody response against Yops. We conclude that Yst plays a major role in the Y enterocolitica- induced diarrhea of the young rabbit and, hence, that it is a major virulence factor in this model. This conclusion may .EE not be valid for the piglet: Robins-Browne et al. (46) tested a 0 pair of phenotypically Yst+and Yst- strains in gnotobiotic o x piglets and did not observe a clear difference in the occurrence of diarrhea. The discrepancy with our observations 0 could result from the animal model difference. It could also 0 n result from the type of strains used: the phenotypically negative strain presumably carried a silent or poorly expressed yst gene (lOa, 11), and one cannot exclude that this gene was reactived in vivo. Finally, our results are also somewhat contradictory to the observation by O'Loughlin et 1 4 7 9 11 14 17 21 24 al. (35) that the Y enterocolitica-induced diarrhea is not caused by an active intestinal secretion. We do not see any B Day simple explanation for this discrepancy. E S According to our observations, diarrhea markedly increased the bacterial shedding by the rabbits. Yst seems thus Co to be an important factor for the in vivo persistence and the 'B dissemination of the bacteria. Taking into account that the rabbit model displays a number of similarities with human Y enterocolitica infection *CM (i6, 24, 34, 35, 38), we infer that Yst could also be involved in acute enterocolitis in the young child. This conclusion is in as perfect agreement with the observation that all the serotypes that are pathogenic for humans possess the yst gene while the nonpathogenic serotypes do not (11). This assumption could still be reinforced by testing the same pairs of isogenic strains in another relevant animal model such as the mon- 1 4 7 9 11 14 17 21 24 key. The mouse model is probably inadequate, as already noticed by Schiemann (49). 0C Day At variance With the E. coli toxin STI, Yst cannot be detected in vitro at 37°C (3, 37, 45). Moreover, Yst has, so far, not been detected in the intestinal contents of infected 0,O animals (38, 46, 62). Our results, however, clearly demonstrate that Yst must be produced in vivo. This suggests that E the conditions required for the expression of yst are not t reproduced in vitro. This contradiction between in vivo and 0 in vitro production is not unprecedented: the production of C._ cholera toxin (CT) in vitro is much higher at a low temperature (30°C) than at 37°C (40). The expression of the ctxAB operon in vivo is, however, not questioned. The lack of detection of Yst in vivo could simply result from the poor E sensitivity of the suckling mouse assay. Finally, the adherence of enterotoxigenic E. coli strains to 1 4 7 9 11 14 17 21 24 the brush border of epithelial cells of the proximal small intestine is a crucial event in their pathogenesis (for review, Day see reference 53). By analogy, Y enterocolitica presumably FIG. 3. Glolbal clinical evolution of the rabbits during the exper- also requires a colonization factor for the onset of diarrhea. imental infecticin with Y enterocolitica: occurrence of diarrhea (A), The adhesin YadA is a likely candidate. It is a 45-kDa weight loss (B), and mortality (C). 0, group A (control); M, group B pYV-encoded major outer membrane protein (52) forming a (yst mutant); EI, group C (yst+). fibrillar matrix on the surface of Y enterocolitica and Y pseudotuberculosis when they are cultivated at 37°C. It promotes the colonization of mouse intestine (20), and it and that it iis probably a major cause of death in this mediates adhesion of Y enterocolitica to cultured epithelial experimental infection. cells (15). It was also showil to increase the adhesion of Y By contrasit, the incidences of diarrhea, weight loss, and enterocolitica to the rabbit small intestinal tissue and to mortality werre significantly lower in the group of rabbits rabbit ileal brush border membrane vesicles (36). However, inoculated with the yst mutant. The incidences were of the some observations are puzzling for an intestine colonization same order aas those observed in the uninfected control factor. First, YadA adhesion is inhibited by mucus (26, 36). group. Morecver, the rare diarrheic episodes and the two Second, YadA plays a role in the bacterial resistance against deaths that cc curred in the former group were not associated the bactericidal activity of human serum (1). Third, YadA is with excretiorn of Y enterocolitica. Theyst mutant retained, produced by Y pseudotuberculosis (52), which does not however, its capacity to colonize the intestines and to produce Yst (11). Finally, the structure of YadA is quite generate a systemic infection: it was excreted in the feces, different from the enterotoxigenic E. coli pili: in SDS-PAGE, 4276 DELOR AND CORNELIS INF'ECT. IMMUN.

YadA appears as a band migrating with an apparent molec- membrane proteins. J. Clin. Microbiol. 27:1072-1076. ular mass of 200 to 240 kDa. According to Skurnik and 13. Feeley, J. C., J. G. Wells, T. F. Tsai, and N. D. Puhr. 1979. Wolf-Watz (52), this band would represent the fibrillae, Detection of enterotoxigenic and invasive strains of Yersinia made of only four or five intertwined polypeptide chains, enterocolitica. Contrib. Microbiol. Immunol. 5:329-334. 14. Guzman-Verduzco, L.-M., and Y. M. Kupersztoch. 1989. Recti- which are anchored to the outer membrane by the hydro- fication of two Escherichia coli heat-stable enterotoxin allele phobic carboxy-terminal end. This structure is clearly differ- sequences and lack of biological effect of changing the carboxy- ent from that of the fimbriae of the enterotoxigenic E. coli. terminal tyrosine to histidine. Infect. Immun. 57:645-648. Furthermore, the genetic organization encoding the enteric 15. Heesemann, J., and L. Gruter. 1987. Genetic evidence that the fimbriae is totally different from that ofyadA (for review, see outer membrane protein YOP1 of Yersinia enterocolitica medi- reference 53). We feel that it is thus questionable whether ates adherence and phagocytosis resistance to human epithelial YadA-mediated adhesion contributes to Yst-mediated diar- cells. FEMS Microbiol. Lett. 40:37-41. rhea. This point requires further investigations, keeping in 16. Heesemann, J., J. Scroder, and M. Ulrich. 1988. Analysis of the mind that the adhesion factors are generally species specific. class-specific immune response to Yersinia enterocolitica virulence-associated antigens in orogastrically infected rabbits. Mi- crob. Pathog. 5:437-447. ACKNOWLEDGMENTS 17. Inoue, T., K. Okamoto, T. Moriyama, T. Takahashi, K. Shimizu, We thank C. Mauyen, C. Cuthbert, and M. Daix for technical and A. Miyama. 1983. Effect of Yersinia enterocolitica ST on assistance in handling the animals. We also thank K. Kaniga for cyclic guanosine 3',5'-monophosphate levels in mouse intes- discussions, M.-P. Sory and J.-C. Vanooteghem for a critical tines and cultures cells. Microbiol. Immunol. 27:159-166. reading of the manuscript, and the ICP illustration service for the 18. Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host- artwork. range suicide vector for improving reverse genetics in gram- This work was supported by the Belgian ministry for sciences negative bacteria: inactivation of the blaA gene of Yersinia (Action concertee 86/91-86) and by a grant from the Belgian Fund for enterocolitica. Gene 109:137-141. Medical Research (contract 3.4514.83). I.D. was the recipient of a 19. Kaniga, K., M.-P. Sory, I. Delor, C. Saegerman, J. Limet, and scholarship from the Fonds de Developpement Scientifique (FDS) of G. R. Cornelis. 1992. Monotoring of Yersinia enterocolitica in the University of Louvain. murine and bovine feces on the basis of the chromosomally integrated lux4B marker gene. Appl. Environ. Microbiol. 58: REFERENCES 1024-1026. 1. Balligand, G., Y. Laroche, and G. Cornelis. 1985. Genetic 20. Kapperud, G., E. Namork, M. Skurnik, and T. Nesbakken. 1987. analysis of virulence plasmid from a serogroup 9 Yersinia Plasmid-mediated surface fibrillae of Yersiniapseudotuberculo- enterocolitica strain: role of outer membrane protein P1 in sis and Yersinia enterocolitica: relationship to the outer mem- resistance to human serum and autoagglutination. Infect. Im- brane protein YOP1 and possible importance for pathogenesis. mun. 48:782-786. Infect. Immun. 55:2247-2254. 2. Biot, T., and G. R. Cornelis. 1988. The replication, partition and 21. Kohl, S., J. A. Jacobson, and A. Nahmias. 1976. Yersinia yop regulation of the pYV plasmids are highly conserved in enterocolitica infections in children. J. Pediatr. 89:77-79. Yersinia enterocolitica and Y pseudotuberculosis. J. Gen. Mi- 22. Laird, W. J., and D. C. Cavanaugh. 1980. Correlation of crobiol. 134:1525-1534. autoagglutination and virulence of yersiniae. J. Clin. Microbiol. 3. Boyce, J. M., D. J. Evans, Jr., D. G. Evans, and H. L. DuPont. 11:430-432. 1979. Production of heat-stable, methanol-soluble enterotoxin 23. Lathe, R., J. L. Vilotte, and A. J. Clark. 1987. Plasmid and by Yersinia enterocolitica. Infect. Immun. 25:532-537. bacteriophage vectors for excision of intact inserts. Gene 57: 4. Carter, P. B. 1975. Pathogenicity of Yersinia enterocolitica for 193-201. mice. Infect. Immun. 11:164-170. 24. Lian, C.-J., W. S. Hwang, J. K. Kelly, and C. H. Pai. 1987. 5. China, B., T. Michiels, and G. R. Cornelis. 1990. The pYV Invasiveness of Yersinia enterocolitica lacking the virulence plasmid of Yersinia encodes a lipoprotein YlpA, related to TraT. plasmid: an in-vivo study. J. Med. Microbiol. 24:219-226. Mol. Microbiol. 4:1585-1593. 25. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular 6. Cornelis, G., and E. P. Abraham. 1975. 1-Lactamases from cloning: a laboratory manual, p. 504. Cold Spring Harbor Yersinia enterocolitica. J. Gen. Microbiol. 87:273-284. Laboratory, Cold Spring Harbor, N.Y. 7. Cornelis, G., Y. Laroche, G. Balligand, M.-P. Sory, and G. 26. Mantle, M., L. Basaraba, S. C. Peacock, and D. G. Gall. 1989. Wauters. 1987. Y enterocolitica, a primary model for bacterial Binding of Yersinia enterocolitica to rabbit intestinal brush invasiveness. Rev. Infect. Dis. 9:64-87. border membranes, mucus, and mucin. Infect. Immun. 57:3292- 8. Cornelis, G. R., T. Biot, C. Lambert de Rouvroit, T. Michiels, B. 3299. Mulder, C. Sluiters, M.-P. Sory, M. Van Bouchaute, and J.-C. 27. Marks, M. I., C. H. Pai, L. Lafleur, L. Lackman, and 0. Vanooteghem. 1989. The Yersinia yop regulon. Mol. Microbiol. Hammerberg. 1980. Yersinia enterocolitica gastroenteritis: a 3:1455-1459. prospective study of clinical, bacteriologic and epidemiologic 9. Cornelis, G. R., C. Sluiters, I. Delor, D. Geib, K. Kaniga, C. features. J. Pediatr. 96:26-31. Lambert de Rouvroit, M.-P. Sory, J.-C. Vanooteghem, and T. 28. Marmur, J. 1961. A procedure for the isolation of deoxyribo- Michiels. 1991. ymoA, a Yersinia enterocolitica chromosomal nucleic acid from microorganisms. J. Mol. Biol. 3:208-218. gene modulating the expression of virulence functions. Mol. 29. Marsh, L. J., M. Erfle, and E. J. Wykes. 1984. The pIC plasmid Microbiol. 5:1023-1034. and phage vectors with versatile cloning sites for recombinant 10. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harden. selection by insertional inactivation. Gene 32:481-485. 1972. Test for Escherichia coli enterotoxin using infant mice: 30. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector application in a study of diarrhea in children in Honolulu. J. and its use in construction of insertion mutations: osmoregula- Infect. Dis. 125:407-411. tion of outer membrane proteins and virulence determinants in 10a.Delor, I., and G. R. Cornelis. Unpublished data. Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-2583. 11. Delor, I., A. Kaeckenbeeck, G. Wauters, and G. R. Cornelis. 31. Miyama, A., T. Inoue, K. Okamoto, and J. Yukitake. 1987. 1990. Nucleotide sequence of yst, the Yersinia enterocolitica Biological function of heat-stable enterotoxin produced by Yers- gene encoding the heat-stable enterotoxin, and prevalence of inia enterocolitica. Contrib. Microbiol. Immunol. 9:266-271. the gene among the pathogenic and nonpathogenic yersiniae. 32. Okamoto, K., A. Miyama, T. Takeda, Y. Takeda, and T. Infect. Immun. 58:2983-2988. Miwatani. 1983. Cross-neutralization of heat-stable enterotoxin 12. Derclaye, I., I. Delor, M. Van Bouchaute, P. Moureau, G. activity of enterotoxigenic Escherichia coli and of Yersinia Wauters, and G. R. Cornelis. 1989. Identification of Campylo- enterocolitica. FEMS Microbiol. Lett. 16:85-87. bacter jejuni and C. coli by gel electrophoresis of the outer 33. Okamoto, K., and M. Takahara. 1990. Synthesis of Escherichia VOL. 60, 1992 ROLE OF Y EN7EROCOLITICA Yst TOXIN 4277

coli heat-stable enterotoxin STp as a pre-pro form and role of sinia enterocolitica serotype 0:3 is capable of producing diar- the pro sequence in secretion. J. Bacteriol. 172:5260-5265. rhea in mice. Infect. Immun. 32:571-574. 34. O'Loughlin, E. V., G. Humphreys, I. Dunn, J. Kelly, C. J. Lian, 50. Schiemann, D. A., and J. A. Devenish. 1982. Relationship of C. Pai, and D. G. Gall. 1986. Clinical, morphological and HeLa cell infectivity to biochemical, serological, and virulence biochemical alterations in acute intestinal yersiniosis. Pediatr. characteristics of Yersinia enterocolitica. Infect. Immun. 35: Res. 20:602-608. 497-506. 35. O'Loughlin, E. V., C. H. Pai, and D. G. Gall. 1988. Effect of 51. Seaone, A., and J. M. Garcia Lobo. 1991. Cloning of chromo- acute Yersinia enterocolitica infection on in vivo and in vitro somal P-lactamase genes from Yersinia enterocolitica. J. Gen. small intestinal solute and fluid absorption in the rabbit. Gas- Microbiol. 137:141-146. troenterology 94:664-672. 52. Skurnik, M., and H. Wolf-Watz. 1989. Analysis of the yopA 36. Paerregaard, A., F. Espersen, and M. Skurnik. 1991. Adhesion gene encoding the Yopl virulence determinants of Yersinia spp. of yersiniae to rabbit intestinal constituents: role of outer Mol. Microbiol. 3:517-529. membrane protein YadA and modulation by intestinal mucus. 53. Smyth, C. J., M. Boylan, H. M. Matthews, and D. C. Coleman. Contrib. Microbiol. Immunol. 12:171-175. 1991. Fimbriae of human enterotoxigenic Escherichia coli and 37. Pai, C. H., and V. Mors. 1978. Production of enterotoxin by control of their expression. In E. Z. Ron and S. Rottem (ed.), Yersinia enterocolitica. Infect. Immun. 19:908-911. Microbial surface components and toxins in relation to patho- 38. Pai, C. H., V. Mors, and T. A. Seemayer. 1980. Experimental genesis. Plenum Press, New York. Yersinia enterocolitica enteritis in rabbits. Infect. Immun. 28: 54. Sory, M.-P., P. Hermand, J.-P. Vaerman, and G. R. Cornelis. 328-244. 1990. Oral immunization of mice with a live recombinant 39. Pai, C. H., V. Mors, and S. Toma. 1978. Prevalence of entero- Yersinia enterocolitica 0:9 strain that produces the cholera toxigenicity in human and nonhuman isolates of Yersinia enterotoxin B subunit. Infect. Immun. 58:2420-2428. colitica. Infect. Immun. 22:334-338. 55. Southern, E. M. 1975. Detection of specific sequence among 40. Parsot, C., and J. J. Mekalanos. 1990. Expression of ToxR, the DNA fragments separated by gel electrophoresis. J. Mol. Biol. transcriptional activator of the virulence factors in Vibrio chol- 98:503-517. erae, is modulated by the heat shock response. Proc. Natl. 56. Takao, T., N. Tominaga, and Y. Shimonishi. 1984. Primary Acad. Sci. USA 87:9898-9902. structure of heat-stable enterotoxin produced by Yersinia en- 41. Peabody, D. S., C. L. Andrews, K. W. Escudero, J. H. Devine, terocolitica. Biochem. Biophys. Res. Commun. 125:845-851. T. 0. Baldwin, and D. G. Bear. 1989. A plasmid vector and 57. Takao, T., N. Tominaga, S. Yoshimura, Y. Shimonishi, S. Hara, quantitative techniques for the study of transcription termina- T. Inoue, and A. Miyama. 1985. Isolation, primary structure and tion in Eschenichia coli using bacterial luciferase. Gene 75:289- synthesis of heat-stable enterotoxin produced by Yersinia en- 296. terocolitica. Eur. J. Biochem. 152:199-206. 42. Portnoy, D. A., H. Wolf-Watz, I. Bolin, A. B. Beeder, and S. 58. Tauxe, R. V., J. Vandepitte, G. Wauters, S. M. Martin, V. Falkow. 1984. Characterization of common virulence plasmids Goossens, P. De Mol, R. Van Noyen, and G. Thiers. 1987. in Yersinia species and their role in the expression of outer Yersinia enterocolitica infections and pork: the missing link. membrane proteins. Infect. Immun. 43:108-114. Lancet ii:1129-1132. 43. Rao, M. C., S. Guandalini, W. J. Laird, and M. Field. 1979. 59. Van Noyen, R., R. Selderslaghs, J. Bekaert, and G. Wauters. Effects of heat-stable enterotoxin of Yersinia enterocolitica on 1991. Yersinia enterocolitica and other enteric pathogens in ion transport and cyclic guanosine 3',5'-monophosphate metab- patients with suspected appendicitis. Contrib. Microbiol. Immu- olism in rabbit ileum. Infect. Immun. 26:875-878. nol. 12:265-271. 44. Rasheed, J. K., L. M. Guzman-Verduzco, and Y. M. Kupersz- 60. Verhaegen, J., L. Dancsa, P. Lemmens, M. Janssens, L. Verbist, toch. 1990. Two precursors of the heat-stable enterotoxin of J. Vandepitte, and G. Wauters. 1991. Yersinia enterocolitica Escherichia coli: evidence of extracellular processing. Mol. surveillance in Belgium (1979-1989). Contrib. Microbiol. Immu- Microbiol. 4:265-273. nol. 12:11-16. 45. Robertson, D. C., and N. Amirmozafari. 1988. Nutritional re- 61. Wauters, G. 1979. Carriage of Yersinia enterocolitica serotype 3 quirements and chemical characterization of heat-stable enter- by pigs as a source of human infection. Contrib. Microbiol. otoxins produced by Yersinia enterocolitica, p. 271-284. In N. Immunol. 5:249-252. Ohtomo and R. B. Sack (ed.), Advances in research on cholera 62. Whipp, S. C., H. W. Moon, and N. C. Lyon. 1975. Heat-stable and related diarrhea, vol. 6. KTK Scientific Publishers, Tokyo. Escherichia coli enterotoxin production in vivo. Infect. Immun. 46. Robins-Browne, R. M., S. Tzipori, G. Gonis, J. Hayes, M. 12:240-244. Withers, and J. K. Prpic. 1985. The pathogenesis of Yersinia 63. Yoshimura, S., H. Ikemura, H. Wanatabe, S. Aimoto, Y. Shi- enterocolitica infection in gnotobiotic piglets. J. Med. Micro- monishi, S. Hara, T. Takeda, T. Miwatani, and Y. Takeda. 1985. biol. 19:297-308. Essential structure for full enterotoxigenic activity of heat- 47. Robins-Browne, R. M., C. S. Still, M. D. Miliotis, and H. J. stable enterotoxin produced by enterotoxigenic Escherichia Koornhof. 1979. Mechanism of action of Yersinia enterocolitica coli. FEBS Lett. 181:138-142. enterotoxin. Infect. Immun. 25:680-684. 64. Zink, D. L., J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. 48. Schiemann, D. A. 1980. Isolation of toxigenic Yersinia entero- Vickery, W. D. Roof, and G. A. Donovan. 1980. Plasmid- colitica from retail pork product. J. Food Protect. 43:360-365. mediated tissue invasiveness in Y enterocolitica. Nature (Lon- 49. Schiemann, D. A. 1981. An enterotoxin-negative strain of Yer- don) 283:224-226.