<p>During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease chlamydial protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Understanding the role of CPAF in pathogenesis is hampered because Chlamydia are not genetically tractable organisms. As such, chemical biology approaches were used to confirm CPAF function in vitro and in vivo, and to validate it as a virulence target. Here we report the development of assays, investigation of substrate specificity, and establishment of CPAF as a central virulence factor in chlamydial pathogenesis. A system for the expression and purification of CPAF was developed. An in vitro assay would allow for determination of kinetic parameters and aid in understanding the function of this protease. Two in vitro proteolysis assays, a discontinuous HPLC-based assay and a continuous fluorescence quenching assay, were developed for use in kinetic parameter determination and inhibitor discovery. </p><p>CPAF substrate specificity studies were conducted through the use of alanine scanning, proteomic identification of protease cleavage sites (PICS), and quantitative proteomics. Results from these studies showed that CPAF exhibited a preference for glycine, alanine, and serine in position P1, and valine in position P2' of peptide substrates.</p><p>Additionally, we designed and synthesized a zymogen-derived inhibitor peptide with nanomolar affinity that inhibited CPAF activity in vitro and in vivo. Using this, anti-CPAF peptide, we established CPAF as a virulence factor for chlamydial pathogenesis. Furthermore, CPAF inhibition resulted in degradation of the inclusion vacuole, exposing the bacteria and stimulating bacterial killing, thus CPAF inhibition created an antibacterial effect. CPAF inhibition also leads to the stimulation of innate immune defense activation, namely activation of caspase 1. In addition, CPAF was determined to be inhibited by the natural product salinosporamide A, a variant of omuralide, and the active form of the proteasome inhibitor lactacystin. Salinosporamide A and omuralide offer advantages over peptide therapeutics because of their intrinsic resistance to proteolytic degradation and improved oral bioavailability. Toward that end, progress toward CPAF inhibitor derivates from this natural product scaffold is also presented. Collectively this thesis lends support for CPAF as an antivirulence target for Chlamydia.</p> / Dissertation

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-CPAFwt 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.

<p>The obligate intracellular pathogen, Chlamydia trachomatis, is becoming an ever greater public health threat worldwide. Despite aggressive public health awareness campaigns and treatment with antibiotics, chlamydial infections continue to be the most frequently reported sexually transmitted infection in the United States and the cause of 3% of worldwide blindness. While research into understanding various mechanisms of chlamydial pathogenesis is ongoing, efforts to identify critical protein targets are hampered by the lack of facile genetic manipulation systems available for Chlamydia. Without the ability to perform genetic studies, researchers have employed chemical biology tools to close the gap in understanding how Chlamydia survives and thrives in the host cell.</p><p>Chlamydial protease-like activity factor (CPAF) has been identified as a central virulence factor in chlamydial pathogenesis. Several studies have indicated a role for CPAF-mediated degradation of host proteins in the late stages of infection. CPAF is hypothesized to interfere with myriad host cell processes, including inflammation, cell proliferation, cytoskeletal development, and immunity presentation. However, recent studies have called into question the methods used to previously identify bona fide in vivo CPAF targets, as CPAF has been shown to retain proteolytic activity even in the presence of broad spectrum protease inhibitors. As a result of these new finding, there is a renewed call to carefully identify CPAF substrates using methods that ensure total inhibition of post-lysis proteolysis.</p><p>This dissertation aims to clarify the role of CPAF in chlamydial pathogenesis and to identify mechanisms by which CPAF exhibits substrate specificity. Because enzymes can manifest specificity through kinetic mechanisms, sequence recognition, secondary site substrate binding, or protein structure level specificity, multiple methods of biochemical characterization were employed to distinguish between these modes of specificity. </p><p>Optimized HPLC-based and fluorescence quenching assays were developed and used to investigate the chemical and kinetic mechanism of CPAF proteolysis, as well as to characterize CPAF resistance to broad spectrum protease inhibitors. Peptide library proteomics were designed to probe active site sequence recognition of specific amino acids. Bioinformatic approaches were used to recognize and annotate a cryptic PDZ-like domain in CPAF, which bears strong structural similarity to human epithelial tight junction proteins. Using a new endocervical cellular model of infection, a recently developed C. trachomatis mutant lacking CPAF activity was investigated. Mass spectrometry proteomics analysis was employed to detect differential cleavage of host proteins in endocervical cells infected with CPAF+ and CPAF- strains of C. trachomatis. Lastly, methods for N-terminal labeling and enrichment were adapted for further identifying CPAF substrates in a cellular infection model. The subtiligase system for biotinylation of N-terminal amines was adapted for integration with C. trachomatis infection assays and downstream mass spectrometry proteomics. Ultimately, the dissertation offers clarification of the role of CPAF in chlamydial infection and provides chemical biology tools for further study of protease function in bacterial pathogenesis.</p> / Dissertation

<p><italic>Chlamydia<italic> <italic>trachomatis<italic> remains a highly relevant clinical pathogen as it is the causative agent of the most commonly reported sexually transmitted disease in the western hemisphere, and the most common cause of infectious blindness in the developing world. As an obligate intracellular pathogen, <italic>Chlamydia<italic> employs a vast assay of virulence proteins to hijack and remodel the host cellular machinery to facilitate its growth and dissemination. Besides delivering effector proteins into the host cytoplasm via a conserved type III secretion machinery, Chlamydia encodes components of multiple secretion systems, such as type II and IV. Chapter 3 of this document describes the secretion, processing and localization of two putative autotransporters (Pls1 and Pls2) and their involvement in inclusion expansion.</p><p> </p><p>In recent years, many new chlamydial effector proteins have been described. CPAF (Chlamydial Protease-like Activity Factor) is a secreted serine protease that is emerging as a central virulence protein: it is proposed to play a central role in <italic>Chlamydia<italic> pathogenesis by cleaving proteins involved in antigen-presentation, apoptosis and cytoskeletal re-arrangements. However, the functional significance of CPAF remains elusive due to the lack of specific inhibitors and <italic>Chlamydia<italic> mutants. The body of work presented herein demonstrates that in addition to targeting host proteins, CPAF cleaves a subset of early chlamydial effector proteins, including Inc-proteins that reside on the nascent pathogenic vacuole ("inclusion"). The design and development of a CPAF-specific inhibitory peptide demonstrates that these chlamydial effector proteins are true targets of CPAF. This peptide reversed the cleavage of bacterial targets by CPAF both in an in vitro cleavage assay and during infection, indicating that these effectors are bona fide targets. Inhibition of CPAF activity also revealed that this protease regulates multiple facets of chlamydial pathogenesis. CPAF inhibition in infected epithelial cells led to the complete dismantling of the inclusion, secretion of pro-inflammatory cytokines and engagement of an inflammasome-dependent programmed cell death pathway. While fibroblasts defective in various inflammasome components were resistant to <italic>Chlamydia<italic>-induced cell death, inclusion integrity and bacterial replication was still compromised upon CPAF inhibition, indicating that loss of inclusion integrity was not a consequence of caspase-1 activation. Overall, these findings revealed that CPAF, in addition to regulating host function, directly modulates the activity of secreted effectors and early Inc-proteins. Furthermore, we establish that CPAF is an essential virulence factor that is required to maintain the integrity of the inclusion and prevent the engagement of innate immune programmed cell death pathways in infected epithelial cells. CPAF activity thus remains a compelling mechanism by which intracellular pathogens employ proteolytic events to modify the host environment.</p> / Dissertation

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial STD agent in the US. This bacterium has a unique biphasic developmental cycle in which the infectious elementary body (EB) infects a host mucosal epithelial cell and differentiates into the replicative form (the reticulate body or RB) within a modified vacuole called an inclusion. The RB later divides and develops back into an EB and is released, perpetuating the infectious cycle. When developing chlamydiae are exposed to unfavorable environmental conditions, they deviate from the normal developmental cycle into a non-infectious but viable state termed persistence. Previous data from our laboratory indicate that i) during C. trachomatis/HSV co-infection, the chlamydiae become persistent and ii) HSV gD interaction with host cell surface is sufficient to induce this response. During viral entry, HSV gD interacts with one of four host co-receptors, one of which is the host adhesion molecule nectin-1. Interestingly, Western blotting demonstrated that nectin-1 is significantly decreased in C. trachomatis-infected HeLa cells. Additional studies indicated that active C. trachomatis replication is required for nectin-1 down-regulation and nectin-1 is likely down-regulated post-translationally. CPAF, a chlamydia-secreted protease, is responsible for degrading several host proteins. Both in vivo experiments using CPAF-specific chemical inhibitors and cell-free cleavage assays using recombinant CPAF indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Further studies suggest that nectin-1 is the most likely candidate involved in triggering HSV-induced chlamydial persistence. Co-infection experiments using nectin-1-specific HSV-1 mutants suggest that nectin-1 is, indeed, required for persistence induction. Additional studies in single co-receptor-expressing CHO cells demonstrate that, despite the fact that HSV-1 enters both HVEM- and nectin-1-expressing cells, viral co-infection reduces chlamydial infectivity only in the CHO-nectin-1 cell line. These data confirm that HSV/nectin-1 interaction is sufficient for chlamydial persistence induction. Although nectin-1 ligation is known to activate Cdc42, pull-down assays indicate that Cdc42 is not activated in co-infected HeLa cells. Taken together, these data suggest that: i) HSV gD-nectin-1 binding activates a novel host epithelial cell pathway that restricts chlamydial development and ii) the chlamydiae may degrade nectin-1 to evade this inhibitory host response.

Chlamydien sind obligat intrazelluläre Bakterien, die sich in einem membranumschlossenen Kompartiment namens Inklusion vermehren. Nach Infektion fragmentiert der Golgi-Apparat der Wirtszelle in kleine Membranstapel. Dies verbessert die Aufnahme von Sphingolipiden und ist deshalb für die chlamydiale Vermehrung essentiell. Die infektionsinduzierte Golgi-Fragmentierung geschieht nach Spaltung des Golgi-Matrix-Proteins Golgin-84. In dieser Arbeit konnte, durch den Vergleich mit bekannten Substraten und Inhibitorstudien, die chlamydiale Protease CPAF (Chlamydia protease-like activity factor) als das Enzym identifiziert werden, das diese Spaltung induziert, abhängig von der Anwesenheit zweier Rab-Proteine, Rab6 und Rab11, die den zellulären Vesikeltransport kontrollieren und zur Inklusion rekrutiert werden. Die Fragmentierung des Golgi-Apparates verhinderte dessen Relokalisierung während der Zellpolarisierung nach Einbringen eines migratorischen Stimulus. Sowohl infizierte als auch Golgin-84-depletierte Zellen migrierten langsamer und randomisiert in einem Motilitätsassay. Die Relokalisierung des Golgi-Apparates konnte durch seine Stabilisierung mittels WEHD oder Rab-Depletion wieder gewonnen werden, was die Zellmotilität teilweise wieder herstellte. Darüber hinaus konnte gezeigt werden, dass die Infektion außer der Golgi-Reorientierung die Signaltransduktion durch GTPasen beeinflusst. Die Aktivität von Cdc42 in infizierten Zellen war erhöht und die Interaktionen mit vielen ihrer Effektoren laut quantitativer Massenspektrometrie stark verändert. Die Ergebnisse dieser Arbeit zeigen, dass CPAF die für Chlamydien lebenswichtige Golgin-84 Prozessierung und Fragmentierung des Golgi-Apparates auslöst. Dies verringert die Mobilität der Wirtszelle, vor allem da der Golgi-Apparat während der Polarisierung nicht mehr ausgerichtet werden kann, des Weiteren durch Modulierung der Protein-Protein-Interaktionen von Cdc42. / Chlamydia are obligate intracellular human pathogens that proliferate inside a membrane-bound compartment called the inclusion. In infected cells, the Golgi apparatus is fragmented into small ministacks that are aligned around the inclusion. This facilitates uptake of host cell sphingolipids and is essential for chlamydial development. Infection-induced Golgi fragmentation happens after processing of the Golgi matrix protein golgin-84. This work could, via comparison with well-known substrates and inhibitor studies, identify the chlamydial protease CPAF (Chlamydia protease-like activity factor) as the enzyme accountable for this cleavage. Golgi Fragmentation depended on two Rab proteins, Rab6 and Rab11, which control vesicle transport and are recruited to the Chlamydia inclusion. As a consequence of Golgi fragmentation, cells lost the capacity to reorient the Golgi apparatus during polarization after a migratory stimulus. Both infected and golgin-84 depleted cells with a permanently fragmented Golgi apparatus displayed decelerated and furthermore randomized migration in a motility assay. Relocalization of the Golgi apparatus could be restored via stabilizing WEHD treatment or Rab depletion which partly rescued cell motility. Moreover, it could be shown that migration signaling via small GTPases was influenced by Chlamydia infection. Infected cells exhibited activation of the small polarity GTPase Cdc42. Numerous interactions with downstream effectors were strongly altered in infected cells according to quantitative mass spectrometry. Particularly, the binding of Cdc42 to migration-associated effectors was decreased. The results of this work show that CPAF, by processing of golgin-84, induces Golgi fragmentation which is vitally important for Chlamydia. This disturbs host cell motility because the Golgi apparatus cannot be reoriented during polarization and, additionally, via the modulation of protein-protein-interactions of Cdc42.