Protease CPAF Research Articles

The unexpected effect of the protease CPAF from Chlamydia trachomatisis to increase the global synthesis of the host proteins

Abstract MHC-I molecules are involved in the predisposition to several immune diseases. Reactive arthritis (ReA) is strongly linked with Chlamydia trachomatisinfection and HLA-B*27. However, how the MHC-I can contribute to the pathology is not well understood. Chlamydiais an intracellular pathogen and one of its main pathogenic factors is CPAF. This enzyme is secreted to the cytosol of the host during infection and there are no data regarding its global effect on the protein degradation and how could affect the ligands presented by MHC-I molecules. T-REx-293 cells stably transfected with CPAF were induced with doxycycline and its effects on several known substrates were assayed by Western blot. Moreover, its unknown effects on the global proteome were evaluated by label-free quantitation (LFQ) using a Q-Exactive plus mass spectrometer. Additionally, MHC-I complexes were purified by immunoprecipitation and the eluted peptides were analyzed by MS. CPAF is highly active degrading known substrates such as Vimentin and RFX5, but also showed an unexpected and profound effect on the cellular proteome increasing the expression of many proteins with a wide range of functions, mostly related to transcription and translation pathways. More than 3000 MHC-I-restricted peptides were identified showing that the peptidome is altered when CPAF is induced, increasing the diversity and altering the physicochemical properties of the peptides. As conclusion, CPAF deeply alters the host proteome contributing to alter the peptide supply of the antigen processing pathway which affect the composition of the MHC-I-associated immunopeptidome. These data suggest that the generation of “new ligands” could be a new pathogenic mechanism of C. trachomatisin ReA. Supported by grants Fondecyt 11170969 and Fondequip EQM190142 from ANID, Chile.

A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF.

Urogenital Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted diseases globally. Untreated C. trachomatis infections can ascend to the upper genital tract and establish a series of severe complications. Previous studies using C3−/− and C5−/− mice models demonstrated that C3-independent activation of C5 occurred during C. trachomatis infection. However, the mechanism of how chlamydial infection activates C5 in the absence of C3 has yet to be elucidated. To delineate interactions between C5 and chlamydial infection, cleavage products in a co-incubation system containing purified human C5 and C. trachomatis-HeLa229 cell lysates were analyzed, and a novel cleavage pattern of C5 activation induced by C. trachomatis infection was identified. C5 was cleaved efficiently at the previously unidentified site K970, but was cleaved poorly at site R751. C5b was modified to C5bCt, which later formed C5bCt-9, which had enhanced lytic ability compared with C5b-9. The chlamydial serine protease CPAF contributed to C3-independent C5 activation during C. trachomatis infection. Nafamostat mesylate, a serine protease inhibitor with a good safety profile, had a strong inhibitory effect on C5 activation induced by chlamydial infection. These discoveries reveal the mechanism of C3-independent C5 activation induced by chlamydial infection, and furthermore provide a potential therapeutic target and drug for preventing tubal fibrosis caused by chlamydial infection.

Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity.

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.

Neutralizing antichlamydial activity of complement by chlamydia-secreted protease CPAF

Ascending infection by sexually transmitted Chlamydia trachomatis is required for chlamydial induction of tubal pathology. To achieve ascension, the C. trachomatis organisms may have to spread from cell to cell, which inevitably exposes the organisms to extracellular mucosal effectors such as complement factors that are known to possess strong antichlamydial activities. Here, we report that the chlamydia-secreted protease CPAF efficiently neutralized complement factor C3-dependent antichlamydial activity. The neutralization was dependent on the proteolytic activity of CPAF and correlated with the CPAF-mediated degradation of complement factor C3 and factor B. As a result, CPAF preferentially inhibited the alternative complement activation pathway. The significance and limitation of these observations were discussed.

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.

Induction and inhibition of CPAF activity during analysis of Chlamydia-infected cells.

Studies of the chlamydial protease CPAF have been complicated by difficulties in distinguishing bona fide intracellular proteolysis from in vitro proteolysis. This confounding issue has been attributed to CPAF activity in lysates from Chlamydia-infected cells. We compared three methods that have been used to inhibit in vitro CPAF-mediated proteolysis: (1) pre-treatment of infected cells with the inhibitor clasto-lactacystin, (2) direct cell lysis in 8 M urea and (3) direct lysis in hot 1% SDS buffer. We identified a number of experimental conditions that reduce the effectiveness of each method in preventing CPAF activity during lysate preparation. The amount of in vitro proteolysis in a lysate was variable and depended on factors such as the specific substrate and the time in the intracellular infection. Additionally, we demonstrated for the first time that artifactual CPAF activity is induced before cell lysis by standard cell detachment methods, including trypsinization. Protein analysis of Chlamydia-infected cells therefore requires precautions to inhibit CPAF activity during both cell detachment and lysate preparation, followed by verification that the cell lysates do not contain residual CPAF activity. These concerns about artifactual proteolysis extend beyond studies of CPAF function because they have the potential to affect the analyses of host and chlamydial proteins from Chlamydia-infected cells.

The Chlamydia protease CPAF: caution, precautions and function.

Recent biochemical and genetic studies have called into question many of the functions proposed for the chlamydial protease CPAF (Zhong et al ., 2001). In 2012, we demonstrated that the proteolysis of multiple reported substrates was occurring during the preparation of lysates from Chlamydia infected cells rather than in intact cells (Chen et al ., 2012). The recent report by Snavely et al . of a CPAF null mutant now provides genetic evidence that CPAF is not necessary for many Chlamydia induced phenotypes nor for a successful intracellular infection (Bavoil & Byrne, 2014; Snavely et al ., 2014). The proteolysis observed when analyzing Chlamydia infected cells must be carefully interpreted because lysates prepared under standard conditions contain CPAF proteolytic activity (Chen et al ., 2012). In general, proteolysis during lysate preparation is not a problem for protein analysis because proteases in cell lysates are typically inactive on ice and are readily inhibited with a protease inhibitor cocktail. However, CPAF is unusual because it remains active in cell lysates at 4 °C and is resistant to most protease inhibitors (Zhong et al ., 2000; Jorgensen et al ., 2011; Chen et al ., 2012). As an unfortunate consequence of this ongoing enzymatic activity, CPAF cleaves or degrades numerous host proteins during lysate preparation from infected cells. Multiple studies have reported that CPAF mediated proteolysis of specific host proteins begins at about 10–20 h postinfection (hpi) (Dong et al ., 2004; Kumar & Valdivia, 2008; Christian et al ., 2011). However, we did not detect the published proteolysis of 11 host proteins, even as late as 48 hpi, if we inhibited CPAF activity at the time of lysate preparation (Chen et al ., 2012). These doubts about the proteolysis of proposed CPAF targets challenge …

Analysis of CPAF mutants: new functions, new questions (The ins and outs of a chlamydial protease)

The role of the chlamydial protease CPAF, previously described as a secreted serine protease processing a wealth of host and chlamydial proteins to promote chlamydial intracellular growth, has recently been questioned by studies from the groups of Tan and Sütterlin, who demonstrated that the reported proteolysis of almost a dozen substrates by CPAF occurred during preparation of cell lysates rather than in intact cells. Valdivia et al. have now compared near-isogenic pairs of CPAF-deficient and secretion-deficient mutants of Chlamydia trachomatis and their wild-type parent. Their report, published in this issue of Pathogens and Disease, is a landmark study in the emerging era of Chlamydia genetics. The results of Tan and Sütterlin are confirmed with a few additions. While CPAF's role in pathogenesis is diminished considerably from these studies, CPAF remains an important factor in chlamydial biology as (1) CPAF mutants produce less infectious yield than wild type; and (2) CPAF is responsible for proteolytic cleavage of vimentin and LAP-1, but only after lysis of the inclusion membrane, not upon CPAF secretion to the cytosol. Here, we briefly review the evidence in support of CPAF's active secretion from the mid-to-late inclusion and conclude that new experimentation to establish whether or not CPAF is actively secreted should precede any new investigation of CPAF's cellular activities during mid-to-late development.

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.

Multinucleation during C. trachomatis infections is caused by the contribution of two effector pathways.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C.trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S(-) or CPAF(-) C.trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C.trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin-associated protein-1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.

Chlamydia trachomatis Outer Membrane Complex Protein B (OmcB) Is Processed by the Protease CPAF

We previously reported that the Chlamydia trachomatis outer membrane complex protein B (OmcB) was partially processed in Chlamydia-infected cells. We have now confirmed that the OmcB processing occurred inside live cells during chlamydial infection and was not due to proteolysis during sample harvesting. OmcB processing was preceded by the generation of active CPAF, a serine protease known to be able to cross the inner membrane via a Sec-dependent pathway, suggesting that active CPAF is available for processing OmcB in the periplasm. In a cell-free system, CPAF activity is both necessary and sufficient for processing OmcB. Both depletion of CPAF from Chlamydia-infected cell lysates with a CPAF-specific antibody and blocking CPAF activity with a CPAF-specific inhibitory peptide removed the OmcB processing ability of the lysates. A highly purified wild-type CPAF but not a catalytic residue-substituted mutant CPAF was sufficient for processing OmcB. Most importantly, in chlamydial culture, inhibition of CPAF with a specific inhibitory peptide blocked OmcB processing and reduced the recovery of infectious organisms. Thus, we have identified OmcB as a novel authentic target for the putative chlamydial virulence factor CPAF, which should facilitate our understanding of the roles of CPAF in chlamydial biology and pathogenesis.

The Chlamydia Protease CPAF Regulates Host and Bacterial Proteins to Maintain Pathogen Vacuole Integrity and Promote Virulence

The Chlamydia Protease CPAF Regulates Host and Bacterial Proteins to Maintain Pathogen Vacuole Integrity and Promote Virulence

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.