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Characterization of CPAF critical residues and secretion during Chlamydia trachomatis infection.

ABSTRACT: CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF's tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.

SUBMITTER: Yang Z

PROVIDER: S-EPMC4432759 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Characterization of CPAF critical residues and secretion during Chlamydia trachomatis infection.

Yang Zhangsheng Z Tang Lingli L Sun Xin X Chai Jijie J Zhong Guangming G

Infection and immunity 20150316 6

CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were d ...[more]

PMID: 25776755

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Project description: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 NF?B-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.

Project description:Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

Project description:UnlabelledThe Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia.ImportanceThis study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.

Dataset Information

Characterization of CPAF critical residues and secretion during Chlamydia trachomatis infection.

Publications

Characterization of CPAF critical residues and secretion during Chlamydia trachomatis infection.

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