Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA.

Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.

Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts.

IMPORTANCE: Chronic or repeated infection of the female upper genital tract by C. trachomatis can lead to severe fibrotic sequelae, including tubal factor infertility and ectopic pregnancy. However, the molecular mechanisms underlying this effect are unclear. In this report, we define a transcriptional program specific to C. trachomatis infection of the upper genital tract, identifying tissue-specific induction of host YAP-a pro-fibrotic transcriptional cofactor-as a potential driver of infection-mediated fibrotic gene expression. Furthermore, we show that infected endocervical epithelial cells stimulate collagen production by fibroblasts and implicate chlamydial induction of YAP in this effect. Our results define a mechanism by which infection mediates tissue-level fibrotic pathology via paracrine signaling and identify YAP as a potential therapeutic target for the prevention of Chlamydia-associated scarring of the female genital tract.

The role of infected epithelial cells in Chlamydia-associated fibrosis.

Ocular, genital, and anogenital infection by the obligate intracellular pathogen Chlamydia trachomatis have been consistently associated with scar-forming sequelae. In cases of chronic or repeated infection of the female genital tract, infection-associated fibrosis of the fallopian tubes can result in ectopic pregnancy or infertility. In light of this urgent concern to public health, the underlying mechanism of C. trachomatis-associated scarring is a topic of ongoing study. Fibrosis is understood to be an outcome of persistent injury and/or dysregulated wound healing, in which an aberrantly activated myofibroblast population mediates hypertrophic remodeling of the basement membrane via deposition of collagens and other components of the extracellular matrix, as well as induction of epithelial cell proliferation via growth factor signaling. Initial study of infection-associated immune cell recruitment and pro-inflammatory signaling have suggested the cellular paradigm of chlamydial pathogenesis, wherein inflammation-associated tissue damage and fibrosis are the indirect result of an immune response to the pathogen initiated by host epithelial cells. However, recent work has revealed more direct routes by which C. trachomatis may induce scarring, such as infection-associated induction of growth factor signaling and pro-fibrotic remodeling of the extracellular matrix. Additionally, C. trachomatis infection has been shown to induce an epithelial-to-mesenchymal transition in host epithelial cells, prompting transdifferentiation into a myofibroblast-like phenotype. In this review, we summarize the field's current understanding of Chlamydia-associated fibrosis, reviewing key new findings and identifying opportunities for further research.

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion.

Introduction: The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors. Methods: To identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting. Results: RNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation - which was instead sensitive to inhibition of Abl. Discussion: Our results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP.

The iron-dependent repressor YtgR is a tryptophan-dependent attenuator of the trpRBA operon in Chlamydia trachomatis.

The trp operon of Chlamydia trachomatis is organized differently from other model bacteria. It contains trpR, an intergenic region (IGR), and the biosynthetic trpB and trpA open-reading frames. TrpR is a tryptophan-dependent repressor that regulates the major promoter (PtrpR), while the IGR harbors an alternative promoter (PtrpBA) and an operator sequence for the iron-dependent repressor YtgR to regulate trpBA expression. Here, we report that YtgR repression at PtrpBA is also dependent on tryptophan by regulating YtgR levels through a rare triple-tryptophan motif (WWW) in the YtgCR precursor. Inhibiting translation during tryptophan limitation at the WWW motif subsequently promotes Rho-independent transcription termination of ytgR, thereby de-repressing PtrpBA. Thus, YtgR represents an alternative strategy to attenuate trpBA expression, expanding the repertoire for trp operon attenuation beyond TrpL- and TRAP-mediated mechanisms described in other bacteria. Furthermore, repurposing the iron-dependent repressor YtgR underscores the fundamental importance of maintaining tryptophan-dependent attenuation of the trpRBA operon.

A bipartite iron-dependent transcriptional regulation of the tryptophan salvage pathway in Chlamydia trachomatis.

During infection, pathogens are starved of essential nutrients such as iron and tryptophan by host immune effectors. Without conserved global stress response regulators, how the obligate intracellular bacterium Chlamydia trachomatis arrives at a physiologically similar 'persistent' state in response to starvation of either nutrient remains unclear. Here, we report on the iron-dependent regulation of the trpRBA tryptophan salvage pathway in C. trachomatis. Iron starvation specifically induces trpBA expression from a novel promoter element within an intergenic region flanked by trpR and trpB. YtgR, the only known iron-dependent regulator in Chlamydia, can bind to the trpRBA intergenic region upstream of the alternative trpBA promoter to repress transcription. Simultaneously, YtgR binding promotes the termination of transcripts from the primary promoter upstream of trpR. This is the first description of an iron-dependent mechanism regulating prokaryotic tryptophan biosynthesis that may indicate the existence of novel approaches to gene regulation and stress response in Chlamydia.

Genomewide Transcriptional Responses of Iron-Starved Chlamydia trachomatis Reveal Prioritization of Metabolic Precursor Synthesis over Protein Translation.

Iron is essential for growth and development of Chlamydia. Its long-term starvation in cultured mammalian cells leads to production of aberrant noninfectious chlamydial forms, also known as persistence. Immediate transcriptional responses to iron limitation have not been characterized, leaving a knowledge gap of how Chlamydia regulates its response to changes in iron availability. We used the fast-chelating agent 2,2'-bipyridyl (BPDL) to homogeneously starve Chlamydia trachomatis serovar L2 of iron, starting at 6 or 12 h postinfection. Immediate transcriptional responses were monitored after only 3 or 6 h of BPDL treatment, well before formation of aberrant Chlamydia. The first genomewide transcriptional response of C. trachomatis to iron starvation was subsequently determined utilizing RNA sequencing. Only 7% and 8% of the genome were differentially expressed in response to iron starvation at the early and middle stages of development, respectively. Biological pathway analysis revealed an overarching theme. Synthesis of macromolecular precursors (deoxynucleotides, amino acids, charged tRNAs, and acetyl coenzyme A [acetyl-CoA]) was upregulated, while energy-expensive processes (ABC transport and translation) were downregulated. A large fraction of differentially downregulated genes are involved in translation, including those encoding ribosome assembly and initiation and termination factors, which could be analogous to the translation downregulation triggered by stress in other prokaryotes during stringent responses. Additionally, transcriptional upregulation of DNA repair, oxidative stress, and tryptophan salvage genes reveals a possible coordination of responses to multiple antimicrobial and immunological insults. These responses of replicative-phase Chlamydia to iron starvation indicate a prioritization of survival over replication, enabling the pathogen to "stock the pantry" with ingredients needed for rapid growth once optimal iron levels are restored. IMPORTANCE By utilizing an experimental approach that monitors the immediate global response of Chlamydia trachomatis to iron starvation, clues to long-standing issues in Chlamydia biology are revealed, including how Chlamydia adapts to this stress. We determined that this pathogen initiates a transcriptional program that prioritizes replenishment of nutrient stores over replication, possibly in preparation for rapid growth once optimal iron levels are restored. Transcription of genes for biosynthesis of metabolic precursors was generally upregulated, while those involved in multiple steps of translation were downregulated. We also observed an increase in transcription of genes involved in DNA repair and neutralizing oxidative stress, indicating that Chlamydia employs an "all-or-nothing" strategy. Its small genome limits its ability to tailor a specific response to a particular stress. Therefore, the "all-or-nothing" strategy may be the most efficient way of surviving within the host, where the pathogen likely encounters multiple simultaneous immunological and nutritional insults.

Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.

Ironing Out the Unconventional Mechanisms of Iron Acquisition and Gene Regulation in Chlamydia.

The obligate intracellular pathogen Chlamydia trachomatis, along with its close species relatives, is known to be strictly dependent upon the availability of iron. Deprivation of iron in vitro induces an aberrant morphological phenotype termed "persistence." This persistent phenotype develops in response to various immunological and nutritional insults and may contribute to the development of sub-acute Chlamydia-associated chronic diseases in susceptible populations. Given the importance of iron to Chlamydia, relatively little is understood about its acquisition and its role in gene regulation in comparison to other iron-dependent bacteria. Analysis of the genome sequences of a variety of chlamydial species hinted at the involvement of unconventional mechanisms, being that Chlamydia lack many conventional systems of iron homeostasis that are highly conserved in other bacteria. Herein we detail past and current research regarding chlamydial iron biology in an attempt to provide context to the rapid progress of the field in recent years. We aim to highlight recent discoveries and innovations that illuminate the strategies involved in chlamydial iron homeostasis, including the vesicular mode of acquiring iron from the intracellular environment, and the identification of a putative iron-dependent transcriptional regulator that is synthesized as a fusion with a ABC-type transporter subunit. These recent findings, along with the noted absence of iron-related homologs, indicate that Chlamydia have evolved atypical approaches to the problem of iron homeostasis, reinvigorating research into the iron biology of this pathogen.

Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron-dependent transcriptional repressor.

The regulation of iron homeostasis is essential for most organisms, because iron is required for a variety of conserved biochemical processes, yet can be toxic at high concentrations. Upon experiencing iron starvation in vitro, the obligate intracellular human pathogen Chlamydia trachomatis exhibits elevated expression of a putative iron-transport system encoded by the ytg operon. The third component of the ytg operon, CT069 (YtgCR), encodes a protein with two distinct domains: a membrane-anchored metal ion permease and a diphtheria toxin repressor (DtxR)-like transcriptional repressor. In this report, we demonstrate that the C-terminal domain of CT069 (YtgR) serves as an iron-dependent autorepressor of the ytg operon. Moreover, the nascent full-length metal permease-transcriptional repressor protein was processed during the course of infection, and heterologously when expressed in Escherichia coli. The products produced by heterologous cleavage in E. coli were functional in the repression of a reporter gene downstream of a putative YtgR operator. We report a bona fide mechanism of iron-dependent regulation of transcription in Chlamydia. Moreover, the unusual membrane permease-DNA-binding polypeptide fusion configuration was found in several bacteria. Therefore, the DNA-binding capability and liberation of the YtgR domain from a membrane-anchored permease in C. trachomatis could represent a previously uncharacterized mechanism for prokaryotic regulation of iron-homeostasis.

An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis.

Iron is an essential cofactor in a number of critical biochemical reactions, and as such, its acquisition, storage, and metabolism is highly regulated in most organisms. The obligate intracellular bacterium, Chlamydia trachomatis experiences a developmental arrest when iron within the host is depleted. The nature of the iron starvation response in Chlamydia is relatively uncharacterized because of the likely inefficient method of iron depletion, which currently relies on the compound deferoxamine mesylate (DFO). Inefficient induction of the iron starvation response precludes the identification of iron-regulated genes. This report evaluated DFO with another iron chelator, 2,2'-bipyridyl (Bpdl) and presented a systematic comparison of the two across a range of criteria. We demonstrate that the membrane permeable Bpdl was superior to DFO in the inhibition of chlamydia development, the induction of aberrant morphology, and the induction of an iron starvation transcriptional response in both host and bacteria. Furthermore, iron starvation using Bpdl identified the periplasmic iron-binding protein-encoding ytgA gene as iron-responsive. Overall, the data present a compelling argument for the use of Bpdl, rather than DFO, in future iron starvation studies of chlamydia and other intracellular bacteria.

Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.