Yersinia enterocolitica is an enteropathogenic bacterium that causes a broad spectrum of diseases ranging from gastrointestinal disorders such as enteritis, enterocolitis and mesenteric lymphadenitis, as well as systemic manifestations including reactive arthritis, erythema nodosum, uveitis and septicemia. The pathogenicity of Y. enterocolitica depends on a virulence plasmid (pYV) that encodes a type three secretion system (TTSS), secreted Yersinia outer proteins (Yops) as well as a major outer membrane protein, Yersinia adhesin A (YadA). In particular, the yop virulon enables Yersinia to overcome the innate immune system of their host and to survive in the lymphoid tissues. The TTSS enables extracellularly located yersiniae to translocate at least six toxic effector proteins (Yops) directly into the cytosol of the host cells where YopE, YopT and YopO cause destruction of the actin microfilament structures by modifying small Rho GTPases YopH is a protein tyrosine phosphatase, which acts on various eukaryotic proteins such as the focal adhesion kinase, paxillin and p130Cas thereby inhibiting YopM is distributed within the nucleus of host cells and stimulates the activity of two kinases. YopP affects eukaryotic cells by interfering with NF-#B signaling pathways via inhibition of the activation of I#B kinase and MAPK kinases resulting in reduced cytokine production and apoptosis in macrophages. The chromosomally encoded outer membrane protein invasin (Inv) Inv appears to be important in the early phase of the infection by promoting intestinal translocation of the pathogen and colonization of Peyer’s patches.
Immunity against Y. enterocolitica is based on innate and acquired host defence mechanisms. Y. enterocolitica appears to be located exclusively extracellularly in host tissues and Y. enterocolitica induces abscesses and few granuloma-like lesions in infected tissue. Yersinia are taken up by M cells, which occur within the follicle-associated epithelial of the Peyer´s patches.and immune responses in the mucosa-associated lymphoid tissue are initiated as the Peyer´s patches are important components of the afferent limb of this system. One day after M cell invasion by Y. enterocolitica, small microabscesses consisting of polymorphonuclear leukocytes and extracellularly located Yersinia can be detected in Peyer´s patch tissue. The type of inflammatory responses induced in Peyer´s patches can be considered as a net-effect resulting from two contrary events: Yersinia spp. obviously trigger the recruitment of inflammatory cells such as PMN which are normally absent from Peyer´s patches. This could be a result of the function of e.g., Yersinia invasin protein. Recent work demonstrated that invasin, via binding to host cell #1 integrins, can trigger activation of nuclear factor kappa B (NF-#B) in epithelial cells which leads to expression of various genes such as Interleukin (IL)-8. On the other hand, translocation of e.g., YopP/J via the type III secretion apparatus may suppress expression of proinflammatory genes via inhibition of NF-#B activation and may induce apoptosis in macrophages. Histological and immunohistological analyses suggest that innate host defence mechanisms including PMN and macrophages are involved in control of Yersinia in Peyer´s patches. After replication in Peyer´s patches enteric Yersinia spp. disseminate via the lymphatics and, possibly via the blood stream, to the mesenteric lymph nodes, spleen, liver, lungs and peripheral lymph nodes. It is well established that Yersinia infection leads to strong T cell responses including CD4 and CD8 T cells, and that the T cells are involved in control of Yersinia. In fact, mice deficient for T cells are unable to control the pathogen, and therefore develop chronic progressive and fatal infection. The role of cytokines in yersiniosis has been extensively studied in mice by comparison of mouse strains that are relatively susceptible (e.g., BALB/c) or resistant (e.g., C57BL/6) to Yersinia infection. One reason for this differential susceptibility of mice is their different ability to mount TH1 responses upon Yersinia infection including production of IFN-#. Yersinia-resistant C57BL/6 mice can be rendered Yersinia-susceptible by in vivo neutralizing IFN-# with monoclonal antibodies.Vice versa, Yersinia-susceptible BALB/c mice can be rendered resistant by treatment with IFN-#. More recent work with cytokine- or cytokine receptor-decificent mice clearly demonstrated that the cytokines TNF-#, IL-12, IL-18 and IFN-#, and the transcription factor interferon consensus sequence binding protein are all essential for control of Yersinia infection.
Results/Project Status
Modulation of gene expression in epithelial cells
Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Y. enterocolitica wild type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv) which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22,283 probesets (14,239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-#B-regulated factors such as cytokines and adhesion molecules.Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-#B regulated genes at 2 h postinfection while other pYV-encoded factors repressed host cell genes at 4 and 8 h postinfection. Chromosomally encoded factors of Yersinia other than Inv induced expression of genes known to be induced by TGF-# receptor signaling. These genes were also repressed by pYV-encoded factors. Further studies are in progress to identify the bacterial factors which trigger and suppress this response, respectively. In addition the biological function of this response will be investigated.
Only a few host genes were found exclusively induced by pYV-encoded factors. We hypothesized that some of these genes may contribute to pYV-mediated silencing of host cells. For instance, the glucocorticoid induced leucine zipper (GILZ), a member of the leucine zipper family was observed to be induced by Yersinia in epithelial cells. GILZ was described to be induced by different stimuli such as dexamethsone or IL-10 or by IL-2 deprivation in different cell types such as T cells and macrophages. GILZ expression protects T cells from apoptosis and prevents NF-#B activation. We demonstrated that wildtype Yersinia which carry the pYV plasmid (pYV+) upregulated GILZ mRNA and protein expression in epithelial cells. Yersinia induced GILZ expression was dependent on the presence of YopT, which cleaves the membrane-bound form of Rho GTPases and affects the actin cytoskeleton. Similarly, inhibitors of Rho GTPases such as Clostridium difficile toxin B induced GILZ protein expression. These data indicated that inactivation of Rho GTPases mediated GILZ induction. Transient overexpression of RhoA and RhoB inhibited GILZ expression at a transcriptional and posttranscriptional level. Overexpression of GILZ in HeLa cells inhibited NF-#B activation as well as IL-8 promoter activity induced by Yersinia invasin. YopT in cooperation with YopP inhibited NF-#B activation and IL-8 secretion. Therefore we conclude that YopT mediated inactivation of Rho GTPases may contribute to Yersinia inhibition of IL-8 secretion by induction of GILZ. This indicates a novel mechanism by which Yersinia may suppress host response. Taken together Yersinia mediates gene silencing in epithelial cells by two distinct mechanisms: (i) A direct inhibition by YopP and (ii) an indirect “autocrine loop” mediated by YopT induced GILZ. Currently we investigating GILZ expression upon Yersinia infection and the possible role of GILZ during infection.
Modulation of gene expression upon Yersinia infection in vivo in C57/BL/6
To identify effector mechanisms which might be crucial for clearance of Yersinia enterocolitica infection we investigated
gene expression upon Yersinia infection in early phase of infection comprising cells which express the cell surface marker CD11b+, namely macrophages, granulocytes, dendritic cells and NK cells. Prior to and 1 and 3 days after systemical infection CD11b+ cells were purified by MACS separation and the distribution of the above indicated cell populations was determined. In addition gene expression analyses were performed to determine changes in gene expression profiles upon Yersinia infection. 538 probe sets were more than three-fold differentially expressed upon Yersinia one day or three postinfection. This immune response is characterized by induction of a high number of chemokines, cytokines and interferon induced genes. We compared the gene expression profile generated in acute yersiniosis with the gene expression profiles generated in a IBD model using IL-2-/- deficient mice kept under specific pathogen free (SPF) - conditions which develop in contrast to germfree IL-2-/- mice or SPF IL-2-/- mice developing colitis. The comparison revealed that although using cells from different organs/cell population such as CD11b+ cells from spleen (yersiniosis) showed a high overlap of genes upregulated in both systems indicating that the host response due to acute and chronic inflammation is highly comparable and that a tight regulation is crucial to avoid chronic inflammation. Studies on TLR signaling which leads subsequently to induction of different cytokines showed that induction of host response is crucial to overcome infection. Since the induced genes might represent important mechanisms which may be crucial to overcome Yersinia infection and especially could be crucial to evade immune evasion mechanisms a high number of these genes (approximately 200) were cloned in cooperation with Bernd Korn, RZPD Heidelberg and Jan Mollenhauer into expression vector to generate stable cell lines which will be used in future studies to generally elucidate the role of these genes on various cell processes such as e.g cell proliferation, cell viability as well as to investigate which of these genes might overcome immune evasion mechanims by Yersinia or other pathogens. Having finshed the cloning process currently the cell lines are generated which overexpress these genes.
Role of cytokines for clearance of infection and modulation of gene expression
Cytokines such as IFN-#, IL-10 and TNF-##play an important role for the clearance of Yersinia infection. Thus, IFN-##receptor and TNF-receptor deficient mice show an increased susceptiblitiy against Yersinia while IL-10 deficient mice are more resistant against Yersinia. We wanted to know which genes are modulated upon Yersinia infection in CD11b+ cells in these mice and whether we can define effector mechanisms which could be responsible for the different susceptibility. Using these approach we defined that 53 probe sets in TNF-R, 61 probe sets in IL-10, and 103 probesets in IFN-#R deficient mice were differentially expressed. Real-time PCR confirmed for several genes the microarray data. However only 16 were no longer regulated at all upon infection in cytokine/cytokine-receptor deficient mice. Such genes were exclusively found in IFN-#R deficient mice indicating that these genes could be essential for the increased susceptibility of IFN-#R-/- for Yersinia infection in vivo.
Role of virulence factors for clearance of infection and modulation of gene expression
To investigate how Yops modulate gene expression in vivo C57BL/6 mice were infected with wildtype yersiniae and a YopH or YopP mutant for 1 and 3 days. While YopH leads to a reduced bacterial number in the spleen compared to wildtype, bacterial number upon infection with a YopP mutant is rather similar or even higher in early phase of infection. Infection with YopH deficient strain leads to a decreased influx of granulocytes into the spleen. In addition a distinct group of genes can be defined (e.g. some chemokines) which are lower expressed upon infection with YopH deficient Yersinia. This effect however, seems to be primarily due to the lower bacterial number in the spleen and gene expression can be restored by an increased infection dose leading to a bacterial load similar to infection with wildtype Yersinia. Interestingly a second big group of genes is found where gene expression is for a broad range of a different bacterial load similarly expressed upon infection with wildtype Yersinia and the YopH mutant strain. Only a few genes were found where gene expression might be YopH specifically affected probably by indirect effects. In contrast to in vitro studies addressing the interaction of macrophages with Yersinia, YopP had no or a low impact on genes involved in proinflammatory response or apoptosis in vivo.
Outlook
Having defined the gene expression in vivo upon Yersinia infection we will focus on several genes which might be important for clearance of infection, such as some IFN induced genes. Therefore we use several approaches including in vitro studies using cell lines overexpressing these genes, localization of these genes in in vivo infection by in-situ hybridization and generation of knockout-mice, and infection of knockout-mice models.
Lit.: 1. Koeberle M, Manncke B , Krejci J, Grass GA, Gaentzsch P, Reineke K, Steffen Müller S, Asselin-Labat ML, Pallardy M, Cornelis GR, Wolf-Watz H, Zumbihl R, Autenrieth IB, Erwin Bohn. 2005. Targeting of RhoGTPAses by YopT leads to induction of GILZ which contributes to NF-kB inhibition in epithelial cells. Manuscript submitted. 2. Schmid Y, Grassl G., Oliver T. Buehler OT, Skurnik M, Autenrieth IB, Bohn E..2004. Yersinia enterocolitica YadA activates IL-8 by engagement of MAP kinase cascades. Infect. Immun. 172:6780-6789. 3. Bohn E*, Müller S*, Lauber J, Geffers R, Speer N, Spieth C, Krejci J, Manncke B, Buer J, Zell A, Autenrieh IB. 2004. Gene expression patterns of epithelial cell by pathogenicity factors of Yersinia enterocolitica. Cell. Microbiol. 6:129-41. 4. Bohn, E., I. B. Autenrieth. 2004. Immune response to Yersinia. In: Molecular and Cellular Biology of Yersina. Ed. E. Carniel, J. Hinnebush. Horizon Scientific Press/Calster.
