Chlamydia-secreted protease CPAF degrades host antimicrobial peptides

Chlamydia trachomatis infection in the lower genital tract, if untreated, can ascend to the upper genital tract, potentially leading to complications such as tubal factor infertility. The ascension involves cell-to-cell spreading, which may require C. trachomatis organisms to overcome mucosal extracellular effectors such as antimicrobial peptides. We found that among the 8 antimicrobial peptides tested, the cathelicidin LL-37 that is produced by both urogenital epithelial cells and the recruited neutrophils possessed a most potent antichlamydial activity. Interestingly, this antichlamydial activity was completely inhibited by CPAF, a C. trachomatis-secreted serine protease. The inhibition was dependent on CPAF's proteolytic activity. CPAF selectively degraded LL-37 and other antimicrobial peptides with an antichlamydial activity. CPAF is known to secrete into and accumulate in the infected host cell cytoplasm at the late stage of chlamydial intracellular growth and may be released to confront the extracellular antimicrobial peptides before the intra-inclusion organisms are exposed to extracellular environments during host cell lysis and chlamydial spreading. Thus, the finding that CPAF selectively targets host antimicrobial peptides that possess antichlamydial activities for proteolysis suggests that CPAF may contribute to C. trachomatis pathogenicity by aiding in ascending infection.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease

Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious inflammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of pro-apoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia-associated cell damage, and inflammation.     

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to na?ve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells.     

Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens

Attaching and effacing (A/E) pathogens such as enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) cause serious global health problems. These bacteria colonize the gastrointestinal system, attach to intestinal epithelial cells, efface (collapse) infected cell microvilli and cause overt diarrhoea that may ultimately result in death of the host. Although pathogenically induced diarrhoea is a significant global health issue, the molecular mechanisms that underlie this disease remain largely unknown. A natural murine infection model, employing the A/E pathogen Citrobacter rodentium, has been helpful in studying the diseases in vivo. C. rodentium colonize the colon at high levels, attach to colonocytes, efface microvilli and cause hyperplasia and inflammation in infected mice. As the disease progresses, the mice develop a diarrhoea-like phenotype. Aquaporin (AQP) water channels have been proposed to play a role in the normal dehydration of faecal contents. Here we examine whether C. rodentium infection may alter AQP localization in colonocytes. We demonstrate that during infection, AQP2 and AQP3 are mislocalized from their normal location along cell membranes to the cell cytoplasm. The change in localization of these proteins correlates with the diarrhoea-like phenotype present in infected mice. Mice that recover from the infection at 28-35 days post inoculum regain their normal membrane AQP localization. The altered localization of AQPs is partially dependent on the bacterial type III effector proteins EspF and EspG. We conclude that altered AQP localization may be a contributing factor to diarrhoea during bacterial infection.     

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA,cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.     

Inhibition of the NF-kappaB pathway by varicella-zoster virus in vitro and in human epidermal cells in vivo

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Using human cellular DNA microarrays, we found that many nuclear factor kappa B (NF-kappaB)-responsive genes were down-regulated in VZV-infected fibroblasts, suggesting that VZV infection inhibited the NF-kappaB pathway. The activation of this pathway causes a cellular antiviral response, including the production of alpha/beta interferon, cytokines, and other proteins that restrict viral infection. In these experiments, we demonstrated that VZV interferes with NF-kappaB activation in cultured fibroblasts and in differentiated epidermal cells in skin xenografts of SCIDhu mice infected in vivo. VZV infection of fibroblasts caused a transient nuclear translocation of p50 and p65, the canonical NF-kappaB family members. In a process that was dependent upon the presence of infectious VZV, these proteins rapidly became sequestered in the cytoplasm of VZV-infected cells. Exclusion of NF-kappaB proteins from nuclei was associated with the continued presence of IkappaBalpha, which binds p50 and p65 and prevents their nuclear accumulation. IkappaBalpha levels did not diminish even though the protein became phosphorylated and ubiquitinated, as determined based on detection of the characteristic high-molecular-weight form of the protein, and the 26S proteasome remained functional in VZV-infected cells. VZV infection also inhibited the characteristic degradation of IkappaBalpha that is induced by exposure of fibroblasts to tumor necrosis factor alpha. As expected, herpes simplex virus 1 caused the persistent nuclear translocation of NF-kappaB proteins, which has been shown to facilitate its replication, whereas VZV infection progressed without persistent NF-kappaB nuclear localization. We suggest that VZV has evolved a mechanism to limit host cell antiviral defenses by sequestering NF-kappaB proteins in the cytoplasm, a strategy that appears to be unique among the herpesviruses.