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

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

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

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

A Functional Slow Recycling Pathway of Transferrin is Required for Growth of Chlamydia

An inhibitor of host cell lysophospholipid acyltransferase, an enzyme involved in lipid metabolism blocked growth of the obligate intracellular pathogen Chlamydia through its action on the transport of transferrin (Tf) via the slow pathway of recycling. A detailed characterization of this inhibition revealed that Tf accumulated in vesicles positive for Rab11, with a concomitant reduction in the level of Tf found within the transport intermediate Rab4/11 hybrid vesicles. The net result was the failure to be recycled to the plasma membrane. In chlamydiae-infected cells, the Tf-containing Rab11-positive vesicles were typically found intimately associated with the inclusion, and treatment with the inhibitor caused their accumulation, suggesting that the timely progression and completion of Tf recycling was necessary for proper chlamydial growth. Growth inhibition by the compound could be negated by the simple removal of the Tf-containing fraction of the serum, a further indication that accumulation of Tf around the chlamydial inclusion was deleterious to the pathogen. Thus, it appears that manipulating the slow recycling pathway can have biological consequences for Chlamydia and implies the need to regulate carefully the interaction of the inclusion with this host trafficking pathway

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

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