Alternatively Activated Macrophages Are Host Cells for Chlamydia trachomatis and Reverse Anti-chlamydial Classically Activated Macrophages

The obligate intracellular pathogen Chlamydia trachomatis (Ctr) is the causative agent of the most common form of sexually transmitted disease in the United States. Genital infections with C. trachomatis can lead to inflammatory tissue damage followed by scarring and tissue remodeling during wound healing. Extensive scarring can lead to ectopic pregnancy or infertility. Classically activated macrophages (CA mϕ), with their anti-microbial effector mechanisms, are known to be involved in acute inflammatory processes during the course of infection. In contrast, alternatively activated macrophages (AA mϕ) contribute to tissue repair at sites of wound healing, and have reduced bactericidal functions. They are present during infection, and thus potentially can provide a growth niche for C. trachomatis during a course of infection. To address this question, macrophages derived from CD14-positive monocytes magnetically isolated from peripheral blood mononuclear cells (PBMC) were treated with interferon-γ or interleukin-4 to produce CA mϕ or AA mϕ, respectively. Confocal microscopy of chlamydial inclusions and quantification of infectious yields revealed better pathogen growth and development in AA mϕ than CA mϕ, which correlated with the reduced expression of indoleamine 2,3-dioxygenase, a known anti-chlamydial effector of the host. Furthermore, AA mϕ stained strongly for transferrin receptor and secreted higher amounts of anti-inflammatory interleukin-10 compared to CA mϕ, characteristics that indicate its suitability as host to C. trachomatis. CA, AA, and resting mϕ were infected with Ctr serovar L2. The data suggest that IL-10 produced by infected AA mϕ attenuated the anti-chlamydial function of CA mϕ with growth recovery observed in infected CA mϕ in the presence of infected, but not mock-infected AA mϕ. This could be related to our observation that IL-10 treatment of infected CA mϕ promoted better chlamydial growth. Thus, in addition to serving as an additional niche, AA mϕ might also serve as a means to modulate the immediate environment by attenuating the anti-chlamydial functions of nearby CA mϕ in a manner that could involve IL-10 produced by infected AA mϕ

Machine Learning and Metabolic Model Guided CRISPRi Reveals a Central Role for Phosphoglycerate Mutase in \u3ci\u3eChlamydia trachomatis\u3c/i\u3e Persistence

Upon nutrient starvation, Chlamydia trachomatis serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence is an adaptive response or lack of it. To understand that transcriptomics data were collected for nutrientsufficient and nutrient-starved CTL. Applying machine learning approaches on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions without having any global stress regulator. This indicated that CTL’s stress response is due to lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (iCTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized iCTL278, we observed phosphoglycerate mutase (pgm) regulates the entry of CTL to the persistence. Later, pgm was found to have the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown of pgm and tryptophan starvation experiments revealed the importance of this gene in inducing persistence. Hence, this work, for the first time, introduced thermodynamics and enzyme-cost as tools to gain deeper understanding on CTL persistence

The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector

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

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

IntroductionThe 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.MethodsTo 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.ResultsRNA 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.DiscussionOur 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

Bacterial subversion of host actin dynamics at the plasma membrane

Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion

The analysis of iron-responsive transcriptional regulation in the intracellular human pathogen, Chlamydia trachomatis

The host-pathogen interaction is dictated by the competition for nutrients. Iron is a rare, but critical component for several conserved biochemical reactions, and as such, is at the forefront in the competition between host and pathogen. The obligate intracellular human pathogen, Chlamydia trachomatis, enters an alternative growth mode when iron is depleted within the host. Termed persistence, this growth mode is characterized by the arrest of development until suitable conditions arise within the host cell, and is likely an important evolutionary mechanism for chronic infection that has been linked to multiple sequelae of infection in vivo. Despite the relevance of persistence in human disease, the sensory and response mechanisms that trigger this alternative growth mode are not understood. The research presented in this report demonstrates a new, robust model for the iron-restriction of intracellular-dwelling C. trachomatis. The model was used to elicit uniform populations of persistent Chlamydiae in vitro, which allowed accurate analysis of differential transcription under iron-starvation conditions. From these studies, the gene ytgA, which is predicted to function as part of an ABC-metal transport complex, was elevated upon low-iron availability. The identification of the trans- and cis-regulatory factors responsible for the modulation of ytgA transcription was attempted. Our research indicates that the ytgC, which exists in an operon with ytgA, encodes a genetic fusion between a predicted membrane channel forming polypeptide (N-terminus) and a metal-dependent DNA binding polypeptide (C-terminus), which we have termed C-YtgC. C-YtgC recognized the IGR upstream of ytgA in an iron-dependent manner in vitro, providing a molecular mechanism for the regulation of ytgA under iron-limiting conditions. Two regions containing cis-regulatory elements within the IGR of ytgA were identified for this binding. Additional preliminary evidence suggests a mechanism for the liberation of the C-YtgC polypeptide from the membrane channel portion of the protein. In summary, the research presented in this report has gained significant advances in the field of Chlamydia iron-biology. The search for a robust iron-restriction model has implicated that the chlamydial iron source may be cytosolic, rather than endocytic. Furthermore, the characterization of a novel trans-regulatory factor suggests that Chlamydia may employ a sophisticated regulatory network for maintenance of iron-homeostasis. Continued research into the mechanisms of iron acquisition is warranted, considering the strong link between low-iron availability and chronic infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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