Immunomodulation in mice by experimental infection with Yersinia enterocolitica

Intraperitoneal infection of mice with two strains of Yersinia enterocolitica resulted in an inflammatory response and immunomodulation which appeared to be related to the invasive properties of the bacteria. The primary antibody response to sheep erythrocytes was enhanced by noninvasive cultures of Y. enterocolitica (serotype O:4-33 grown at 22 C and at 37 C, and serotype O:3 grown at 37 C), when given at the same time or two days after the antigen (invasiveness was tested on HeLa cells). In contrast, invasive cultures of serotype O:3 grown at 22 C, injected three days before the antigen suppressed the antibody response; enhancement was caused by these cultures only when given on the day of immunization. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed by invasive cultures of Y. enterocolitica. These data indicate that the temperature of growth as well as some serotype-linked factors play a role in immunomodulation by Y. enterocolitica.     

Nucleotide-binding oligomerization domain 2 (Nod2) is dispensable for the innate immune responses of macrophages against Yersinia enterocolitica

Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for muramyl dipeptide, a component of bacterial peptidoglycan. In this study, we have examined whether Nod2 mediates the immune response of macrophages against Yersinia enterocolitica. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Nod2-deficient mice and were infected with various strains of Y. enterocolitica. ELISA showed that the production of IL-6 and TNF-α in BMDMs infected with Y. enterocolitica was not affected by the Nod2 deficiency. iNOS mRNA expression was induced in both WT and Nod2-deficienct BMDMs in response to Y. enterocolitica, beginning 2 h after infection. Nitric oxide (NO) production by Y. enterocolitica did not differ between WT and Nod2-deficient BMDMs. Western blot analysis revealed that Y. enterocolitica induces activation of NF-κB, p38, and ERK MAPK through a Nod2-independent pathway. Neither LDH release by Y. enterocolitica nor the phagocytic activity of the macrophages was altered by Nod2 deficiency. An in vivo experiment showed that bacterial clearance ability and production of IL-6 and KC in serum were comparable in WT and Nod2-deficient mice infected with Y. enterocolitica. These findings suggest that Nod2 may not be critical for initiating the innate immune response of macrophages against Yersinia infection.     

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFbeta-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-gamma and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-beta1 and IL-4 production by BALB/c mice and to an increase in the IFN-gamma levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.     

Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow-derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis-infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.     

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

Production of autoantibodies associated with polyclonal activation in Yersinia enterocolitica O: 8-infected mice

Polyclonal lymphocyte stimulation is one of the immunomodulatory mechanisms induced by arthritogenic pathogens. In this study we examined the polyclonal activation potential of a virulent strain of Y. enterocolitica serotype O: 8 (WA 2707(+)) and its plasmidless isogenic pair (WA 2707(-)). SPF Swiss mice were infected intragastrically and spleen cells were obtained on days 7, 14, 21, 28, 35 and 42 after infection. The number of cells secreting nonspecific immunoglobulins of IgG, IgM and IgA isotypes was determined by the ELISPOT technique. The presence of serum-specific antibodies was investigated by ELISA and the presence of autoantibodies by dot-blot assay. Although the patterns of infection of the two bacterial strains were almost the same, only the animals infected with the virulent strain presented clinical anomalies. Neither arthritic nor inflammatory signs were observed in the joints of the infected animals. The greatest activation observed was that of the nonspecific IgM-secreting cells, and their peak of secretion occurred between the 28th and the 42nd day after infection, for both strains of Y. enterocolitica O: 8. Only the animals infected with the virulent strain (WA 2707(+)) produced IgG-specific antibodies in the serum, from the 28th day after infection. The serum of animals infected with either strain showed reactivity to all the autologous constituents tested, mainly on the 28th and 42nd day after infection. It was concluded that infection of mice with either the virulent strain of Y. enterocolitica O: 8 or with its plasmidless isogenic pair resulted in the polyclonal activation of the splenic B lymphocytes including some autoreactive clones.     

Identification of rpoE and nadB as host responsive elements of Yersinia enterocolitica

We describe the identification of the Yersinia enterocolitica rpoE gene, which encodes the alternative sigma factor sigmaE, and the divergently transcribed nadB gene, as genes that are expressed during infection of mice. As in Escherichia coli, rpoE of Y. enterocolitica is essential for growth and its expression is autoregulated. Despite the similarities of the rpoE operons of Y. enterocolitica and E. coli, there are considerable differences in the response to extracytoplasmic stress. Unlike in E. coli, sigmaE of Y. enterocolitica is not induced by heat shock or ethanol. Overproduction of the outer membrane protein Ail does not lead to the induction of rpoE expression. However, rpoE expression is induced by osmotic stress.     

Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3

Bacteriophage phiYeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ' gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their beta-galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that phiYeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Recognition of Yersinia enterocolitica multiple strain infection in twin infants using PCR-based DNA fingerprinting

AIMS: Yersinia enterocolitica causes several syndromes in humans. The most common presentation is enterocolitis in children, presenting as fever and diarrhoea. A Y. enterocolitica multiple strain infection in twin infants was investigated. METHODS AND RESULTS: One isolate was recovered from one patient and two morphologically-different isolates were recovered from the other infant. Biochemically, all isolates were identified as Y. enterocolitica group. The genomic DNA from each strain was purified and DNA fingerprinting was performed. The banding patterns observed for Y. enterocolitica isolates 2 and 3, from patients 1 and 2, respectively, were identical when comparing the presence or absence of major bands. However, Y. enterocolitica isolate 1, from patient 1, showed a distinctive banding pattern from isolates 2 and 3. CONCLUSION: The findings indicate that one infant was colonized by more than one strain of Y. enterocolitica, demonstrating that multiple strains can colonize and invade a patient. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of multiple strain infections can be important in diagnosis, treatment and prognosis of Y. enterocolitica infections, as well as in disease epidemiology. The technique described here offers a straightforward method for strain comparison.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.     

PspG, a new member of the Yersinia enterocolitica phage shock protein regulon

The Yersinia enterocolitica phage shock protein (Psp) system is induced when the Ysc type III secretion system is produced or when only the YscC secretin component is synthesized. Some psp null mutants have a growth defect when YscC is produced and a severe virulence defect in animals. The Y. enterocolitica psp locus is made up of two divergently transcribed cistrons, pspF and pspABCDycjXF. pspA operon expression is dependent on RpoN (sigma(54)) and the enhancer-binding protein PspF. Previous data indicated that PspF also controls at least one gene that is not part of the psp locus. In this study we describe the identification of pspG, a new member of the PspF regulon. Predicted RpoN-binding sites upstream of the pspA genes from different bacteria have a common divergence from the consensus sequence, which may be a signature of PspF-dependent promoters. The Y. enterocolitica pspG gene was identified because its promoter also has this signature. Like the pspA operon, pspG is positively regulated by PspF, negatively regulated by PspA, and induced in response to the production of secretins. Purified His(6)-PspF protein specifically interacts with the pspA and pspG control regions. A pspA operon deletion mutant has a growth defect when the YscC secretin is produced and a virulence defect in a mouse model of infection. These phenotypes were exacerbated by a pspG null mutation. Therefore, PspG is the missing component of the Y. enterocolitica Psp regulon that was previously predicted to exist.     

Studies on pathogenicity of Yersinia enterocolitica in mice with diabetes mellitus

In this study, we investigated the colonizing ability as well as the association of Yersinia enterocolitica serotype 0:9 to epithelial cells of the intestinal tract, Peyer's patches, mesenteric lymph nodes, liver, spleen and lungs in Alloxan-induced diabetes mellitus in mice and controls. The results showed that: (a) in diabetic mice the Y. enterocolitica colonizing values were in range of 10(6.5)-10(8.25) CFU/g of feces; (b) maximum colonizing values were found in distal ileum and Peyer's patches and lower in colon; (c) the infection was progressive with dissemination of bacteria in the liver, spleen and lung; (d) in control (non-diabetic) mice, the colonizing values were 10-100 times lower than those found in the diabetic batch; (e) the main histopathological changes noticed, namely ileitis, mesenteric lymphadenitis and septicemia, were presumably induced by high bacterial load in the liver, spleen and lung leading to a septic course of infection as well as toxic effects of heat-stable enterotoxins of Y. enterocolitica (Yst). The results were confirmed by electron microscopy observations. Summing up, these results demonstrate that diabetic mice were more susceptible to Y. enterocolitica cells than normal mice.     

Note: Isolation, characterization and epidemiology of Yersinia enterocolitica from humans and animals

During an 11-year period (1983 to 1994), 51 strains of Yersinia enterocolitica were isolated from humans and animals. Specimens were collected from a total of 3601 sources consisting of 956 patients with enteritis, 300 patients with urinary tract infection, 1564 healthy humans, 510 swine, 38 guinea-pigs, 118 rats and 115 rabbits. Five strains of Y. enterocolitica , bio/serogroups 2/O:9 and 4/O:3, virulence positive, were recovered from patients. Forty-two variants of Y. enterocolitica belonging to pathogenic serogroup O:3, Voges-Proskauer-negative biogroup 3 were recovered from swine, rats and rabbits. The rate of isolation of Y. enterocolitica from diarrhoeal swine was apparently greater than those from healthy swine. The incidence of human infections due to Y. enterocolitica was very low and bioserogroups of isolates were different from the strains which were isolated from animals. There was no evidence to suggest that swine were the source of Y. enterocolitica in humans.