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.     

Targeting of a chlamydial protease impedes intracellular bacterial growth

Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.     

The host adherens junction molecule nectin-1 is degraded by chlamydial protease-like activity factor (CPAF) in Chlamydia trachomatis-infected genital epithelial cells

Nectin-1 is an adhesion protein implicated in the organization of adherens junctions and tight junctions in epithelial cells. Previous studies in our laboratory demonstrated that nectin-1 accumulation was significantly decreased in Chlamydia trachomatis-infected HeLa cells. In the present study, Western blot analyses indicated that nectin-1 down-regulation was C. trachomatis concentration-dependent. The half-life of nectin-1 was also greatly diminished in C. trachomatis-infected cells compared to that observed in mock-infected cells, indicating that nectin-1 was likely down-regulated post-translationally. The chlamydia-secreted protease CPAF is known to degrade several important host proteins; CPAF expression within infected cells correlated with the time-dependent cleavage of nectin-1. Notably, CPAF proteolytic activity is inhibited by lactacystin but not by the proteosome inhibitor MG132. In vivo inhibition experiments demonstrated that nectin-1 down-regulation was blocked by lactacystin exposure. In contrast, MG132 had no effect. Finally, cell-free cleavage assays demonstrated that functional recombinant GST-CPAF(wt) protein degrades nectin-1. This degradation was blocked by lactacystin, as previously observed in vivo. Collectively, these results indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells, a novel strategy that chlamydiae may use to aid their dissemination.     

CPAF: A Chlamydial Protease in Search of an Authentic Substrate

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.     

The secreted protease factor CPAF is responsible for degrading pro-apoptotic BH3-only proteins in Chlamydia trachomatis-infected cells

Chlamydia trachomatis has evolved a profound anti-apoptotic activity that may aid in chlamydial evasion of host defense. The C. trachomatis anti-apoptotic activity has been correlated with blockade of mitochondrial cytochrome c release, inhibition of Bax and Bak activation, and degradation of BH3-only proteins. This study presents evidence that a chlamydia-secreted protease factor designated CPAF is both necessary and sufficient for degrading the BH3-only proteins. When the C. trachomatis-infected cell cytosolic extracts were fractionated by column chromatography, both the CPAF protein and activity elution peaks overlapped with the BH3-only protein degradation activity peak. Depletion of CPAF with a CPAF-specific antibody removed the BH3-only protein degradation activity from the infected cell cytosolic extracts, whereas depletion with control antibodies failed to do so. Notably, recombinant CPAF expressed in bacteria was able to degrade the BH3-only proteins, whereas CPAF mutants similarly prepared from bacteria failed to do so. Finally, bacterium-expressed CPAF also degraded the human BH3-only protein Pumaalpha purified from bacteria. These results demonstrate that CPAF contributes to the chlamydial anti-apoptotic activity by degrading the pro-apoptotic BH3-only Bcl-2 subfamily members.     

Golgi Fragmentation and Sphingomyelin Transport to Chlamydia trachomatis during Penicillin-Induced Persistence Do Not Depend on the Cytosolic Presence of the Chlamydial Protease CPAF

Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.     

CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.     

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.     

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.     

Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds

The obligate intracellular bacterial pathogen Chlamydia trachomatis replicates within a large vacuole or "inclusion" that expands as bacteria multiply but is maintained as an intact organelle. Here, we report that the inclusion is encased in a scaffold of host cytoskeletal structures made up of a network of F-actin and intermediate filaments (IF) that act cooperatively to stabilize the pathogen-containing vacuole. Formation of F-actin at the inclusion was dependent on RhoA, and its disruption led to the disassembly of IFs, loss of inclusion integrity, and leakage of inclusion contents into the host cytoplasm. In addition, IF proteins were processed by the secreted chlamydial protease CPAF to form filamentous structures at the inclusion surface with altered structural properties. We propose that Chlamydia has co-opted the function of F-actin and IFs to stabilize the inclusion with a dynamic, structural scaffold while minimizing the exposure of inclusion contents to cytoplasmic innate immune-surveillance pathways.     

Effector proteins of chlamydiae

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.     

The chlamydial protease CPAF: important or not,important for what?

The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.     

Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)

We previously found that rottlerin, a plant-derived small molecule compound, profoundly inhibited Chlamydia trachomatis growth and blocked sphingolipid trafficking from host cell Golgi into chlamydial inclusions. Since the p38-regulated/activated protein kinase (PRAK) is a known target of rottlerin and is activated in Chlamydia trachomatis-infected cells, we investigated the potential role of this kinase in rottlerin-mediated anti-chlamydial activity. However, we found that a PRAK-specific inhibitor failed to inhibit chlamydial growth, suggesting that the kinase activity of PRAK may not be required for chlamydial intracellular replication. This conclusion was supported by the observation that chlamydial organisms replicated equally well in mouse embryonic fibroblast cells with or without PRAK. Moreover, neither the PRAK inhibitor nor PRAK deficiency altered host sphingolipid trafficking into chlamydial inclusions. Finally, rottlerin maintained its anti-chlamydial activity in PRAK-deficient cells. Together, these observations have demonstrated that PRAK is not required for either the rottlerin-mediated anti-chlamydial activity or rottlerin inhibition of sphingolipid trafficking, suggesting that rottlerin may achieve its inhibitory role by targeting other host factors.     

鹦鹉热嗜衣原体蛋白酶样活性因子(CPAF)原核表达与纯化

应用分子生物学技术,选择鹦鹉热嗜衣原体(Chlamydophila psittaci,C.psittaci,Cps)6BC株的CPAF蛋白的免疫优势区基因,进行构建pGEX6p-2/CPAFm重组质粒与重组菌,使用IPTG诱导重组蛋白的表达并分析诱导温度、诱导剂剂量及诱导时间对蛋白表达的影响.重组蛋白以GST琼脂糖凝胶...