Browsing by Subject "Chlamydia"

The Chlamydiaceae are Gram-negative, obligate intracellular bacteria that are significant pathogens of humans and animals. Intracellularly, the bacteria reside in a membrane-bound vacuole, called the inclusion, from which they manipulate host processes to create a niche optimal for survival and propagation. Acquisition of host-derived lipids is essential for chlamydial growth, yet the source of lipids and mechanisms of trafficking to the inclusion are not well-established. The inclusion avoids interaction with several classical membrane and lipid transport pathways. In a functional genomic screen to identify host modulating chlamydial proteins, our lab identified cytosolic lipid droplets (LDs) as potential target organelles of Chlamydia. LDs are postulated to function in many cellular processes, such as lipid metabolism and transport, membrane trafficking, and cell signaling; therefore, we hypothesized that LDs may be important for Chlamydia pathogenesis as a source of lipids or as a platform for regulating other cellular functions. Here, we characterize the interaction between eukaryotic LDs and the chlamydial inclusion.

We find that LDs are recruited to the Chlamydia inclusion, chlamydial infection disrupts neutral lipid homeostasis, and pharmacological prevention of LD formation inhibits chlamydial replication. Chlamydia produces proteins (Ldas) that localize with LDs in yeast and mammalian cells when transiently expressed and are exported out of the inclusion to peripheral lipid-rich structures during infection. By electron microscopy and live cell imaging, we observe the translocation of intact LDs into the Chlamydia inclusion lumen. Biochemical and microscopic analysis of LDs from infected cells reveals that LD translocation may occur at specialized subregions of the inclusion membrane. The Chlamydia Lda3 protein is implicated in LD tethering to the inclusion membrane, and displacement of the protective coat protein, ADRP, from LD surfaces. This phenomenon could provide access for lipases to the LD core for utilization by the replicating bacteria. Additionally, the functional domains of Lda3 involved in binding to LD and inclusion membranes are identified.

In these studies, we identify eukaryotic lipid droplets (LDs) as a novel target organelle important for Chlamydia pathogenesis and describe a unique mechanism of whole organelle sequestration not previously observed for bacterial pathogens. These results represent a fundamental shift in our understanding of host interactions with the chlamydial inclusion, and may represent a new area for research in the field of cellular microbiology.

Intracellular bacteria such as Chlamydia inhabit a single-membrane vacuolar compartment termed the “inclusion”. Chlamydia inclusions offer an escape from host immune defenses and a dedicated spaces for replication. Viable inclusion membranes must be modified by Chlamydia to sequester nutrients and camouflage themselves from host immune defenses that otherwise sense, contain, or eradicate microbial invaders. C. trachomatis, a human-adapted pathogen responsible for widespread sexually transmitted disease, forms inclusions that are protected from gamma-interferon (IFNγ)-induced cell-autonomous immunity. However, it is largely unknown how the unique architecture of host and bacterial components that localize to inclusion membranes impact chlamydial pathogenesis during IFNγ-stimulated conditions. This dissertation details a novel C. trachomatis secreted effector, the gamma resistance determinant or “GarD”, that shields inclusions from cell-autonomous immunity. Specifically, GarD forms a perimeter on inclusion membranes to prevent attack by the IFNγ-inducible enzyme RNF213, which labels GarD-less inclusions with linear poly-ubiquitin protein chains as a signal for elimination. Therefore, GarD operates as an essential antagonist of RNF213-mediated ubiquitylation and allows C. trachomatis to endure human cell-autonomous defenses. Future and ongoing work considers the mechanics of how GarD, RNF213 and additional bacterial and host cell factors affect pathogenesis of Chlamydia species in mammals. Overall, the novel GarD-RNF213 axis is an important expansion of our knowledge of chlamydial pathogenesis and immune responses to vacuole-residing pathogens.

Chlamydia trachomatis (CT) is the most common bacterial agent of sexually transmitted infection and can cause damaging inflammation of the female reproductive tract. As an obligate intracellular pathogen, CT must exit exhausted host cells in a manner that favors successful dissemination. Epithelial cells infected with CT expel decondensed nuclear chromatin at the conclusion of an infectious cycle, and these ensnare CT particles. Whether these chromatin traps benefit the host or the pathogen is not obvious. The overall goal of this work is to begin discerning between these possibilities by determining how chromatin traps impact CT survival following exit and how traps contribute to CT-induced inflammatory processes.

The obligated intracellular bacterium Chlamydia trachomatis is a pathogen of immense clinical and societal importance with over 1.5 million cases of new infection reported annually in the United States alone. With asymptomatic infection and no acquired immunity, Chlamydia infection often lead to epithelial scaring, ectopic pregnancy and infertility. Worldwide, the pathogen also responsibles for the ocular infection called trachoma that is the leading cause of infectious blindness. The bacteria have a diminutive genome consist of just over 900 genes. In addition, C. trachomatis had been historically refractory toward genetic manipulation. With the bacteria relied upon the host for basic metabolic resources, it is hypothesized that the genes retained in its genome is essential for the infectivity, pathogenesis and life cycle of bacteria. Through bioinformatics tools like sequence similarity networks, we have found that Chlamydia retained a complete set of genes involved in the biosynthesis of menaquinone, a membrane bound electron shuttle essential for cellular respiration. The biosynthesis of menaquinone in C. trachomatis involve the recently discovered futalosine pathway. In this project, we developed a molecular toolkit to examine the individual Chlamydial genes involved in menaquinone biosynthesis for its enzymatic function and essentiality. We employed small molecule inhibitors to chemically probe the function of genes in a cellular infection model and found that certain inhibitors of the menaquinone biosynthesis are effective at inducing inhibitory phenotype. Next, we developed a library of codon-optimized expression plasmid for recombinant expression of the Chlamydial protein in E. coli. We were able to enzymatically characterize and structurally determine the flavin prenyltransferase protein CT220 and its involvement in menaquinone biosynthesis. We also utilized E. coli mutants to test the function of the Chlamydial genes using genetic complementation. Lastly, we were able to improve the processes for plasmid-based mutagenesis of C. trachomatis using the recently developed fluorescent reported allelic exchange mutagenesis tool.

Chlamydia trachomatis is the obligate intracellular pathogen responsible for the most common bacterial sexually transmitted infection worldwide. While our front-line antibiotics have been historically successful in combatting chlamydial infections, emerging issues including treatment failure and chlamydial persistence necessitate the development of new therapeutic approaches. In this work, an enzyme-focused approach was devised to explore two of the intricate survival strategies of C. trachomatis: virulence and energy metabolism. We sought to employ biochemistry, enzymology, and chemical biology tools to interrogate enzyme functions and inform the design of new antichlamydial agents. To these ends, our efforts focused on characterization of the virulence-associated effector protein chlamydial protease-like activity factor (CPAF) and the futalosine pathway for menaquinone biosynthesis. Mechanistic analyses of CPAF zymogen maturation and peptide hydrolysis were performed that collectively classified CPAF as a serine protease with a catalytic tetrad. Analogs of the natural product salinosporamide A were subsequently explored as CPAF inhibitors. The futalosine pathway was discovered to be a source of novel antichlamydial targets through traditional and chemical genetics analyses in a HeLa cell model of chlamydial infection. The foundation was also established for studying a specific pathway enzyme, CT263, in a continuous coupled enzyme assay. Collectively, this dissertation has progressed our knowledge of several enzymes involved in critical processes for chlamydial pathogenicity and viability. The insights gained on a mechanistic level and in the context of chlamydial infection have laid the groundwork for pursuing virulence and energy metabolism enzymes as antichlamydial targets and for developing inhibitors of their activity, which are much-needed resources to combat this extremely prevalent sexually transmitted infection.

Chlamydiae are obligate intracellular bacteria that infect a wide range of animal hosts. For a successful infection, interaction with the host cell by use of the type II (T2), type III (T3), and type V (T5) secretion systems is needed to secure nutrients and subvert the host innate-immune responses. While some the substrates and functions of the T3 and T5 secretion systems are known, a comprehensive understanding of what proteins constitute T2S substrates is largely lacking. Only one protein has been confirmed to be a T2S effector in C. trachomatis, the protease CPAF. In this work, we investigate the role of the T2SS and CPAF during C. trachomatis infection.

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 cells, rather than artifacts of post lysis degradation. Here we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. We identified a mutant in Type II secretion (T2S) that accumulates unprocessed CPAF and two strains with nonsense, loss-of-function mutations in cpa. HeLa cells infected with these mutants did not display cleavage of previously reported CPAF substrates and lysates from HeLa cells infected with either T2S- or CPAF- C. trachomatis did not possess any detectable in vitro CPAF proteolytic activity. CPAF-deficient mutants displayed impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. HeLa cells infected with cpa mutant strains were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, altered NFĸB-dependent gene expression, and resistance to reinfection. However, these observations do not imply that previously identified substrates are not targets of processing by CPAF in infected cells. We provide evidence in live cells that CPAF-mediated protein processing of at least two protein targets, vimentin filaments and the nuclear envelope protein Lamin-associated protein 1 (LAP1), a new CPAF substrate, occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. We postulate 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.

We further show defects in T2S impact bacterial attachment to host cells, intracellular growth, the biochemical properties of glycogen, and lytic exit. We sought to understand of the basis of these phenotypes and identify additional substrates of the T2SS which contribute towards them. We use label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for a comprehensive analysis of proteins present within WT and T2S-defective C. trachomatis bacteria. In combination with immunofluorescence analyses, we present evidence of a defined set of secreted proteins that associate with glycogen in the inclusion during infection and propose this polymer functions in regulating proteins secreted into the inclusion lumen.

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.

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.

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.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and is the leading cause of preventable blindness worldwide. Chlamydia is particularly intriguing from the perspective of cell biology because it is an obligate intracellular pathogen that manipulates host cellular pathways to ensure its proliferation and survival. This is achieved through a significant remodeling of the host cell’s internal architecture from within a membrane-bound vacuole, termed the inclusion. However, given a previous lack of tools to perform genetic analysis, the mechanisms by which Chlamydia induces host cellular changes remained unclear. Here I present genetic and molecular mechanisms of chlamydial manipulation of the host cytoskeleton and organelles. Using a forward genetics screen, InaC was identified as a necessary factor for the assembly of an F-actin structure surrounding the inclusion. InaC associated with the vacuolar membrane where it recruited Golgi-specific ARF-family GTPases. Actin dynamics and ARF GTPases regulate Golgi morphology and positioning within cells, and InaC acted to redistribute the Golgi to surround the Chlamydia inclusion. These findings suggest that Chlamydia places InaC at the inclusion-cytosolic interface to recruit host ARF GTPases and F-actin to form a platform for rearranging intracellular organelles around the inclusion. The inclusion is also surrounded by the intermediate filament vimentin and the chlamydial protease CPAF cleaves vimentin in vitro. CPAF-dependent remodeling of vimentin occurred selectively in late stages of the infection. In living cells, this cleavage occurred only after a loss of inclusion membrane integrity, suggesting that CPAF cleaves intermediate filaments specifically during chlamydial exit of host cells. In summary, I have implemented recent forward and reverse genetic approaches in Chlamydia to reveal how it employs effector proteins to manipulate the internal organization of cells in novel ways.

Host cells are equipped with a robust defense program to defend against intracellular pathogens. The cytokine gamma-interferon (IFNg) activates expression of hundreds of genes that identify intracellular pathogens such as Chlamydia trachomatis and Toxoplasma gondii, target pathogen-containing vacuoles (PVs) with host effector proteins, and destroy the pathogen. In mice, the IFNg-induced defense program targeting Toxoplasma and C. trachomatis is controlled by three Immunity-related GTPase M (Irgm) proteins, which coordinate host effectors to efficiently target and destroy intracellular pathogens. The mechanisms by which Irgm proteins regulate cell-autonomous immunity are not well understood. Human cell-autonomous immunity differs substantially from the mouse system, and while the sole human IRGM ortholog is not involved in cell-autonomous immunity to Toxoplasma or Chlamydia, it has been linked with a variety of inflammatory diseases including Crohn’s disease and sepsis. The mechanism linking polymorphisms in human IRGM with autoimmunity is unclear, but murine Irgm knockout models recapitulate similar phenotypes of hyperinflammation associated with human IRGM. Additionally, mouse and human Irgm proteins have been implicated in similar fundamental cellular processes including autophagy and mitochondrial function. Collectively these findings support the central hypothesis that murine Irgm proteins regulate cellular membrane biology to both coordinate cell-autonomous immunity and regulate inflammation. In order to investigate this hypothesis, I leveraged existing Irgm-/- models as well as our novel pan-Irgm-/- mouse lacking all three Irgm proteins. I demonstrated that pan-Irgm-/- cells are completely defective for the targeting of Toxoplasma gondii with host effectors, while Chlamydia trachomatis remains robustly targeted by most but not all effectors. In spite of the differences in host-directed targeting of these two vacuolar pathogens, pan-Irgm-/- mice were defective for resistance to Toxoplasma and Chlamydia trachomatis both in vitro and in vivo. In pan-Irgm-/- mice infected with Chlamydia trachomatis, adaptive immunity compensated for the defect in cell-autonomous immunity to ultimately clear the infection. Consistent with the hypothesis that Irgm proteins limit inflammation, pan-Irgm-/- mice demonstrated increased inflammation in genital Chlamydia trachomatis infection, and increased inflammation and immunopathology in infection with Chlamydia muridarum. Collectively, my findings shed light on the elusive mechanisms of murine Irgm proteins, clarifying their roles in cell-autonomous immunity, defense against intracellular pathogens in vivo, and regulation of inflammation.

Chlamydia trachomatis is a bacterial pathogen with a large socioeconomic impact: it is the leading cause of preventable blindness worldwide, and the most prevalent sexually transmitted infection in the United States. Despite its importance, relatively little is known about the molecular mechanisms that Chlamydia employs to invade epithelial cells, manipulate the secretory pathway, evade innate immune responses and acquire nutrients from its host. Chlamydia, like many other intracellular pathogens, is known to use a type III secretion mechanism to deliver bacterial effector proteins directly to the host cell cytoplasm. These effectors are thought to be the principle actors involved in co-opting host cell functions. TepP is an effector protein that is pre-loaded into infectious Chlamydia particles, and that is secreted early during infection, but whose function is unknown. We took large-scale, unbiased approaches to identify genes whose transcription is modified during the course of infection in a TepP-dependent manner (microarrays), and proteins that interact with TepP and and/or whose phosphorylation is altered by the absence of TepP (proteomics). We used biochemical techniques, cell biology, and molecular techniques to validate interactions identified using large-scale methods, and to further probe the molecular mechanism underlying these connections. In sum, we have determined that TepP contributes to four major phenotypes: changes in the host cell cytoskeleton, modification of the host cell phosphoproteome, bacterial replication, and interferon-dependent gene activation. We have additionally determined that TepP interacts with the Crk family of host cell adaptor proteins, and the class 1 phosphoinositol-3-kinase (PI3K). Cell lines where the levels or activity of TepP interacting partners were modified by deletion, knockdown, or inhibitors, showed that these host proteins are important for the growth of Chlamydia during infection, but are not required for all TepP-dependent phenotypes. TepP not only interacts with PI3K but also induces its activation during infection. Finally, we have determined that the requirements for phosphorylation of TepP are complex, but that the Src kinases are largely responsible for its phosphorylation. Additionally, Src kinases are required for some TepP-dependent phenotypes, but are not required for the recruitment of TepP-interacting proteins during infection.

Chlamydia trachomatis 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, Chlamydia 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.

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 Chlamydia 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 Chlamydia 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 Chlamydia-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.

The Gram-negative bacteria Chlamydia trachomatis is a common sexually transmitted pathogen that can cause severe sequelae including cause pelvic inflammatory disease and sterility. This obligate intracellular pathogen effectively manipulates host cellular functions by secreting virulence factors across its membrane bound vacuole. Identifying these virulence components and how they help in establishing an environment conducive for bacterial growth is central to understanding chlamydial pathogenesis. This is experimentally challenging due to a lack of tools to perform molecular genetic studies. In the absence of genetic tools, we developed a yeast model system to identify and characterize chlamydial proteins involved in virulence mechanisms. In this study we describe the identification of twenty-eight proteins potentially involved in modulating host cellular functions and the secretion of virulence factors into the host. Since the delivery of virulence proteins by a type three secretion (T3S) system is a critical step for Chlamydia, we identified the proteins that interacted with the T3S apparatus by yeast two-hybrid analysis. We discovered several novel interactions between and determined that the C. trachomatis T3S apparatus displayed a similar architecture to that of other T3S systems. Furthermore with these approaches we identified networks of proteins that interacted with the secretion apparatus including a novel secretion chaperone protein. We characterized Ct260/Mcsc one of the putative secretion and demonstrated that it represents a novel class 1B secretion chaperone protein. Unlike other known chaperones, Mcsc directly interact with a conserved component of the T3S apparatus cytoplasmic domain, CdsQ. These finding represents a novel mechanism by which the secretion chaperone protein Ct260 may increase the secretion efficiency of its effector cargo and may reveal new facets of secretory cargo recognition by T3S systems.

When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of β interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.