衣原体免疫逃逸机制的研究进展

衣原体是一类专性细胞内寄生的原核细胞型微生物,感染人体后可引起多种慢性疾病。衣原体在宿主细胞中复制和持续性存在是其致病的主要原因。近年的研究表明,衣原体通过改变MHC抗原表达、干扰宿主细胞凋亡信号通路等机制以逃逸宿主的免疫清除。     

衣原体调控宿主细胞凋亡的机制研究进展

衣原体是一类专性细胞内寄生的原核微生物,常引起性传播疾病、失明和肺炎等疾病。衣原体可能已经进化出调控宿主细胞的若干机制,通过改变宿主细胞蛋白位置,干扰宿主细胞凋亡信号通路等来抑制细胞凋亡,维持其自身的存活,核转录因子NF-κB也涉及到衣原体感染细胞凋亡抑制;衣原体可以经半胱氨酸蛋白酶依赖途径等机制诱导细胞凋亡,进而感染邻近细胞。     

Recent advances in Chlamydia subversion of host cytoskeletal and membrane trafficking pathways

Chlamydia species are obligate intracellular bacteria that cause sexually transmitted disease, ocular infections and atypical pneumonia. This review highlights recent advances describing the mechanisms by which Chlamydia subvert host cytoskeleton and membrane trafficking pathways to create a replication competent niche.     

Chlamydia attachment to mammalian cells requires protein disulfide isomerase

For Chlamydia, an intracellular pathogen of humans, host cell invasion is obligatory for survival, growth and pathogenesis. At the molecular level, little is known about the binding and entry of Chlamydia into the mammalian host cell. Chlamydia are genetically intractable therefore experimental approaches targeting the host are often necessary. CHO6 is a mutagenized cell line resistant to attachment and infection by Chlamydia. In this study, CHO6 was shown using proteomic methods to have a defect in processing of the leader sequence for protein disulfide isomerase (PDI). Complementation by expression of full-length PDI restored C. trachomatis binding and infectivity in the CHO6 mutant cell line. The cell line was also resistant to diphtheria toxin and required complemented cell-surface PDI for toxin entry. These data demonstrate that native PDI at the cell surface is required for effective chlamydial attachment and infectivity.     

Accumulation of diacylglycerol in the Chlamydia inclusion vacuole: possible role in the inhibition of host cell apoptosis

Intracellular pathogens have developed strategies to survive for extended periods inside their host cells. These include avoidance of host microbicidal effectors, often by sequestration in a protected subcompartment of the host cell. In some cases, the parasites exert also an antiapoptotic effect that prolongs the life of the infected host cell. Chlamydia utilizes both strategies, but the underlying molecular mechanisms are incompletely understood. Comparatively, little is known regarding the effects that Chlamydia exerts on the metabolism and distribution of the host cell lipids. The expression of fluorescently tagged C1 domains revealed that diacylglycerol is greatly accumulated in the immediate vicinity of Chlamydia inclusion vacuoles. The concentrated diacylglycerol recruits protein kinase Cdelta (PKCdelta), a proapoptotic effector, to the immediate vicinity of the vacuole. PKCdelta normally exerts its pro-apoptotic effects at the mitochondria and in the nucleus. We speculate that Chlamydia antagonizes the pro-apoptotic effect of PKCdelta by sequestering the enzyme on the inclusion vacuole away from its conventional target sites. Accordingly, we found that the ectopic expression of a catalytic fragment of PKCdelta that cannot be recruited by the vacuole, because it lacks a functional C1 domain, overcame the anti-apoptotic effect of the bacteria. The scavenging of pro-apoptotic factors may provide a novel mechanism whereby pathogens promote their own survival by extending the life of the host cells they infect.     

Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.     

Chlamydia and programmed cell death

Discordant views regarding host cell death induction by Chlamydia are likely owing to the different methods used for evaluation of apoptosis. Apoptotic and non-apoptotic death owing to both caspase-dependent and -independent activation of the Bax protein occur late in the productive growth cycle. Evidence also suggests that Chlamydia inhibits apoptosis during productive growth as part of its intracellular survival strategy. This is in part owing to proteolytic degradation of the BH3-only family of pro-apoptotic proteins in the mitochondrial pathway. Chlamydia also inhibits apoptosis during persistent growth or in phagocytes, but induces apoptosis in T cells, which suggests that apoptosis has an immunomodulatory role in chlamydial infections. The contribution of apoptosis in disease pathogenesis remains a focus for future research.     

Chlamydial infection induces host cytokinesis failure at abscission

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.     

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.     

Lipid rafts,caveolae, caveolin-1, and entry by Chlamydiae into host cells

Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.     

Silencing or permanent activation: host-cell responses in models of persistent Chlamydia pneumoniae infection

Chlamydia pneumoniae causes respiratory infections. In chronic diseases associated with Chlamydia, such as arteriosclerosis, C. pneumoniae is present in a persistent form, which might participate in pathogenesis of chronic inflammatory disease. To elucidate how these intracellular bacteria modulate host-cells during persistence, we compared the expression pattern of a range of host genes after short (24 h) and long (up to 7 days) times of chlamydia infection in HeLa-cells. One day post infection, in three cell-culture models of persistence, namely treatment with penicillin or IFN-gamma, or iron-depletion, infection induced the genes of CTGF, IL-6, IL-8, IL-11, LIF, EGR-1 and ETV4 in a similar fashion. However, after a longer time, two modes of host-cell reaction emerged that were dependent on the persistence model used. After IFN-gamma and penicillin treatment chlamydia-induced host-cell gene expression was inhibited, while it stayed upregulated in iron-depletion. Human monocytes/macrophages, in which persistence naturally occurs, were additionally investigated: for several genes, UV-inactivated and viable chlamydia caused long-lasting upregulation. Thus, this study reveals (i) the ability of C. pneumoniae to participate in two putative pathomechanisms of persistence, silencing and permanent activation, which might represent different in vivo situations and (ii) a strong dependence on the mode of persistence induction.     

Chlamydia effector proteins and new insights into chlamydial cellular microbiology

Chlamydia and Chlamydophila sp. are highly related obligate intracellular bacterial pathogens that cause sexually transmitted diseases, ocular infections and atypical pneumonias. Relatively little is known about the molecular mechanisms by which Chlamydiae manipulate the mammalian host because they are intractable to genetic manipulation. Studies with heterologous expression systems have revealed a large set of chlamydial proteins that are potentially translocated into the host cytoplasm ('effector' proteins). As new cell biological observations are made and the function of effector proteins begin to be elucidated, a clearer picture of the extent to which Chlamydiae manipulate mammalian cellular processes is beginning to emerge, including the cell cycle, innate immunity, and lipid and membrane transport.     

肺炎衣原体蛋白激酶Cpn0148的克隆表达和定位

目前认为肺炎衣原体(Chlamydia pneumonia, Cpn)除导致呼吸道疾病外, 也是与冠心病相关的重要病原体。作为一种细胞内寄生的病原菌, Cpn激活宿主细胞信号通路, 维护其在细胞内生长代谢, 并导致疾病。肺炎衣原体基因Cpn0148可编码真核细胞样的丝/苏氨酸蛋白激酶, 利用PCR技术扩增全长Cpn0148 ORF, 将其定向插入pGEX-6p原核表达载体, 在大肠杆菌XL-1blue中表达, 测序显示Cpn0148 ORF全长1860 bp, 编码619个氨基酸, 分子量大约70 kD,     

The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly

Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system "plug" proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to αβ-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free αβ-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of αβ-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.     

Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.