作者机构：
[周洲; 吴移谋; 陈丽丽; 李忠玉] 南华大学医学院病原生物学研究所;[谭钢] 南华大学附属第一医院眼科;[钟光明] Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio
摘要：
Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.
摘要：
The pathogenesis of Chlamydia-induced inflammation is poorly understood. pORF5 is the only secreted protein encoded by Chlamydial plasmid. This study aims to investigate the effects of pORF5 on the production of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) and the underlying mechanisms of these effects. THP-1 (a human acute monocytic leukemia cell line) cells were stimulated by pORF5 with or without pretreatment with Natch domain, Leucine-rich repeat and PYD-containing protein 3 (NALP3) siRNA, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) siRNA, cysteine aspartate-specific protease-1 (caspase-1) specific inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. IL-1 beta, IL-18 and caspase-1 expression was detected through both ELISA and qRT-PCR. NALP3 and ASC expression was detected by qRT-PCR. The expression of caspase-1 and phosphorylated-p38 MAPK was detected by western blot analysis. pORF5 induced IL-1 beta, IL-18, caspase-1 and NALP3 inflammasome expression in THP-1 cells. Caspase-1 inhibitor significantly reduced pORF5-induced IL-1 beta and IL-18 expression. The siRNAs for NALP3 inflammasome significantly reduced pORF5-induced IL-1 beta, IL-18 and caspase-1 expression. Furthermore, p38 MAPK inhibitor significantly reduced pORF5-induced IL-1 beta, IL-18, caspase-1 and NALP3 inflammasome expression. pORF5 could induce production of IL-1 beta and IL-18 via NALP3 inflammasome activation and p38MAPK pathway. pORF5 protein might play an important role in Chlamydia pathogenesis. This study provides a new insight into the molecular pathogenesis of Chlamydial diseases.
通讯机构：
[Wu, Yimou] U;Univ South China, Inst Pathobiol, Dept Microbiol, Hengyang, Hunan, Peoples R China.
摘要：
Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.
期刊：
International Journal of Clinical and Experimental Pathology,2014年7(9):5404-5414 ISSN：1936-2625
通讯作者：
Li, Zhongyu
作者机构：
[Li, Zhongyu; Dai, Wenting] Univ South China, Sch Med, Pathogen Biol Inst, Hengyang City 421001, Hunan, Peoples R China.;[Li, Zhongyu] Univ South China, Sch Med, Pathogen Biol Inst, 28 Changshengxi Rd, Hengyang City 421001, Hunan, Peoples R China.
通讯机构：
[Li, Zhongyu] U;Univ South China, Sch Med, Pathogen Biol Inst, 28 Changshengxi Rd, Hengyang City 421001, Hunan, Peoples R China.
关键词：
Chlamydia trachomatis;chaperones;effectors;injectisomes;regulators;type III secretion system
摘要：
Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis.
期刊：
Infection and Immunity,2014年82(12):5327-5335 ISSN：0019-9567
通讯作者：
Zhong, Guangming
作者机构：
[Liu, Yuanjun; Yang, Zhangsheng; Baseman, Joel; Huang, Yumeng; Gong, Siqi; Zhong, Guangming] Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, San Antonio, TX 78229 USA.;[Liu, Quanzhong; Hou, Shuping] Tianjin Med Univ Gen Hosp, Dept Dermatovenereol, Tianjin, Peoples R China.;[Sun, Yina] Tianjin Med Univ, Hosp & Tianjin Inst Endocrinol, Key Lab Hormones & Dev Minist Health, Metab Dis, Tianjin, Peoples R China.;[Li, Zhongyu; Wu, Yimou; Chen, Chaoqun] Univ South China, Dept Microbiol & Immunol, Hengyang, Hunan, Peoples R China.
通讯机构：
[Zhong, Guangming] U;Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, San Antonio, TX 78229 USA.
摘要：
Hydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice.
摘要：
Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.
摘要：
We previously reported that Chlamydia trachomatis hypothetical protein CT311 was secreted out of chlamydial inclusion and into host cell cytosol. We now found that CT311 further entered host cell nucleus at the late stage of infection and continued to accumulate in the nucleus of C. trachomatis-infected cells. When CT311 was expressed via a transgene in mammalian cells, CT311 protein was exclusively detected in the nucleus, suggesting that CT311 by itself is sufficient for nuclear targeting. However, preexisting nuclear CT311 did not affect subsequent chlamydial infection. Using deletion constructs, we mapped a nuclear localization signal sequence of CT311 to residues 21 to 63 ((21)AVEGKPLSRAAQLRERRKDLHVSGKPSPRYALKKRALEAKKNK(63)). This sequence was sufficient for targeting a heterologous protein into mammalian cell nucleus and it contains two independent clusters of basic residues ((34)RERRK(38) and (53)KKRALEAKKNK(63) respectively). Deletion or alanine substitution of the basic residues in either cluster led to loss of nuclear targeting activity, suggesting that both clusters are critical for the nuclear targeting function. These observations have demonstrated that the hypothetical protein CT311 possesses a novel nuclear localization signal sequence with dual modules of basic residues for targeting host cell nucleus during Chlamydia trachomatis infection.
作者机构：
[Wu YiMou; Cao WenJuan; Zhou Zhou; Lu ChunXue; Zhou Hui; Li ZhongYu; Ma KangKang] Univ South China, Pathogen Biol Inst, Hengyang 421001, Peoples R China.;[Xie XiaoBing; Zhou Hui] Hunan Univ Chinese Med, Hosp 1, Dept Lab Med, Changsha 410007, Hunan, Peoples R China.;[Huang QiuLin] Univ South China, Affiliated Hosp 1, Dept Gen Surg, Hengyang 421001, Peoples R China.;[Zhong GuangMing] Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, San Antonio, TX 78229 USA.
通讯机构：
[Li ZhongYu] U;Univ South China, Pathogen Biol Inst, Hengyang 421001, Peoples R China.
关键词：
Chlamydia trachomatis;pORF5 plasmid protein;mitogen-activated protein kinase;proinflammatory cytokines;TLR2
摘要：
Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy. Pathogenesis of infection has been mostly attributed to excessive cytokine production. However, precise mechanisms on how C. trachomatis triggers this production, and which protein(s) stimulate inflammatory cytokines remains unknown. In the present study, the C. trachomatis pORF5 protein induced tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) in dose- and time-dependent manners in the THP-1 human monocyte cell line. We found that intracellular p38/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)/MAPK signaling pathways were required for the induction of TNF-alpha, IL-1 beta and IL-8. Blockade of toll-like receptor 2 (TLR2) signaling reduced induction levels of TNF-alpha, IL-8 and IL-1 beta. We concluded that the C. trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.
摘要：
We previously reported that 5 Chlamydia muridarum antigens reacted with antisera from >90% mice urogenitally infected with C. muridarum and they are TC0660 (ABC transporter or ArtJ), TC0727 (outer membrane complex protein B or OmcB), TC0828 (macrophage infectivity potentiator or MIP), TC0726 (inclusion membrane protein or Inc) & TC0268 (hypothetical protein or HP). The orthologs of these antigens in Chlamydia trachomatis were also highly reactive with antisera from women urogenitally infected with C. trachomatis. In the current study, we evaluated these C. muridarum antigens for their ability to induce protection against a C. muridarum intravaginal challenge infection in mice. We found that only MIP induced the most pronounced protection against C. muridarum infection. The protection correlated well with robust C. muridarum MIP-specific antibody and Th1-dominant T cell responses. The MIP-immunized mice displayed significantly reduced live organism shedding from the lower genital tract and highly attenuated inflammatory pathologies in the upper genital tissues. These results demonstrate that MIP, an immunodominant antigen identified by both human and mouse antisera, may be considered a component of a multi-subunit chlamydial vaccine for inducing protective immunity.
