Browsing by Author "Valdivia, Raphael H"

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.

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.

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.

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.

Human physiology is heavily influenced by the colonization of microbes in the gastrointestinal tract. A major roadblock to understanding this process is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. In order to map bacterial genes, we describe an application of chemical mutagenesis followed by population-based genomic sequencing. We chose to map genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility and also discovered new motility genes that were previously uncharacterized. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host.

This methological advance in gene functional analysis of genetically intractable microbes has enabled us to identify 902 essential genes that are directly responsible for growth and survival. This is achieved by large-scale mutant sequencing analysis. By curating the gene list, we assigned the essentiality of genes to uncharacterized genes as well. These results indicate that the genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains can all be accomplished in bacteria without the development of species-specific molecular genetic tools. Ultimately, this advance helps define the role of genes in complex environments.

To investigate the effect of silent mutations in a gene, we have designed and created thousands of AcGFP codon-variant libraries to determine the relationship between codon usage and protein expression. mRNA structures near the initial start codon regions are prominent factor for determining protein expression level, but variation in sequence beyond the start codon region also importantly modulates expression levels.

Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.

The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.

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.