Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

Pigs are the natural hosts of Chlamydia suis, the only Chlamydia species known to spontaneously acquire homotypic resistance conferred by a class C tetracycline resistance gene. Various susceptibility assays have existed for several years, but there is no widely accepted, standardized assay to determine chlamydial antibiotic susceptibility. In this study, we developed new approaches to determine the in vitro susceptibility of Chlamydia to different antibiotics in view of existing protocols. Specifically, the minimal inhibitory concentration (MIC) is based on a consensus of both inclusion number reduction and alteration of inclusion size and morphology upon antibiotic exposure. In addition to these, we employed a recovery assay, allowing observation of the chlamydial response to drug removal and subsequent recovery, as compared to both continued exposure and to the unexposed control. We propose a simple and fast screening method to detect tetracycline resistant C. suis strains within 2 to 3 days with minimal use of consumables. For proof of principle, we evaluated the susceptibility of three C. suis field strains and the reference strain S45/6 to tetracycline, sulfamethoxazole, and penicillin, antibiotics commonly used to prevent respiratory and gastrointestinal diseases on fattening pig farms. We found that tetracycline sensitive strains can easily be distinguished from resistant strains using the evaluation parameters proposed in this study. Moreover, we report that S45/6 is sensitive to sulfamethoxazole while all evaluated C. suis field strains showed some degree of sulfamethoxazole resistance. Finally, we confirm that Penicillin G induces the chlamydial stress response in all evaluated C. suis strains.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

Background

We previously showed that the burden of Chlamydia pneumoniae in carotid plaques was significantly associated with plaque interleukin (IL)-6, and serum IL-6 and C-reactive protein (CRP), suggesting that infected plaques contribute to systemic inflammatory markers in patients with stroke risk. Since lipoprotein-associated phospholipase A2 (Lp-PLA(2)) mediates inflammation in atherosclerosis, we hypothesized that serum Lp-PLA(2) mass and activity levels and plaque Lp-PLA(2) may be influenced by plaque C. pneumoniae infection.

Methodology/principal findings

Forty-two patients underwent elective carotid endarterectomy. Tissue obtained at surgery was stained by immunohistochemistry for Lp-PLA(2) grade, macrophages, IL-6, C. pneumoniae and CD4+ and CD8+ cells. Serum Lp-PLA(2) activity and mass were measured using the colorimetric activity method (CAM) and ELISA, respectively. Serum homocysteine levels were measured by HPLC. Eleven (26.2%) patients were symptomatic with transient ischemic attacks. There was no correlation between patient risk factors (smoking, coronary artery disease, elevated cholesterol, diabetes, obesity, hypertension and family history of genetic disorders) for atherosclerosis and serum levels or plaque grade for Lp-PLA(2). Plaque Lp-PLA(2) correlated with serum homocysteine levels (p = 0.013), plaque macrophages (p<0.01), and plaque C. pneumoniae (p<0.001), which predominantly infected macrophages, co-localizing with Lp-PLA(2).

Conclusions

The significant association of plaque Lp-PLA(2) with plaque macrophages and C. pneumoniae suggests an interactive role in accelerating inflammation in atherosclerosis. A possible mechanism for C. pneumoniae in the atherogenic process may involve infection of macrophages that induce Lp-PLA(2) production leading to upregulation of inflammatory mediators in plaque tissue. Additional in vitro and in vivo research will be needed to advance our understanding of specific C. pneumoniae and Lp-PLA(2) interactions in atherosclerosis.

Pigs are the natural hosts of Chlamydia suis, the only Chlamydia species known to spontaneously acquire homotypic resistance conferred by a class C tetracycline resistance gene. Various susceptibility assays have existed for several years, but there is no widely accepted, standardized assay to determine chlamydial antibiotic susceptibility. In this study, we developed new approaches to determine the in vitro susceptibility of Chlamydia to different antibiotics in view of existing protocols. Specifically, the minimal inhibitory concentration (MIC) is based on a consensus of both inclusion number reduction and alteration of inclusion size and morphology upon antibiotic exposure. In addition to these, we employed a recovery assay, allowing observation of the chlamydial response to drug removal and subsequent recovery, as compared to both continued exposure and to the unexposed control. We propose a simple and fast screening method to detect tetracycline resistant C. suis strains within 2 to 3 days with minimal use of consumables. For proof of principle, we evaluated the susceptibility of three C. suis field strains and the reference strain S45/6 to tetracycline, sulfamethoxazole, and penicillin, antibiotics commonly used to prevent respiratory and gastrointestinal diseases on fattening pig farms. We found that tetracycline sensitive strains can easily be distinguished from resistant strains using the evaluation parameters proposed in this study. Moreover, we report that S45/6 is sensitive to sulfamethoxazole while all evaluated C. suis field strains showed some degree of sulfamethoxazole resistance. Finally, we confirm that Penicillin G induces the chlamydial stress response in all evaluated C. suis strains.

Chlamydia trachomatis ocular strains cause a blinding disease known as trachoma. These strains rarely cause urogenital infections and are not found in the upper genital tract or rectum. Urogenital strains are responsible for a self-limited conjunctivitis and the sequelae of infertility, ectopic pregnancy, and hemorrhagic proctitis. However, the differential cellular responses that drive these clinically observed disease outcomes are not completely understood. Primary conjunctival, endocervical, and endometrial epithelial and stromal fibroblast cells, HeLa229 cells, and immortalized conjunctival epithelial (HCjE) cells were infected with the ocular A/Har-13 (A) and Ba/Apache-2 (Ba) strains and urogenital D/UW-3 (D) and E/Bour (E) strains. Infection rates, progeny production, and cytokine/chemokine secretion levels were evaluated in comparison with those in uninfected cells. All strain types infected all cell types with similar levels of efficacy and development. However, progeny production levels differed among primary cells: Ba produced significantly more progeny than E in endocervical and endometrial fibroblasts, while A progeny were less abundant than E progeny. C.trachomatis infection of primary epithelial cells elicited an increase in pro- and anti-inflammatory mediators compared to levels in uninfected cells, but there were no significant differences by strain type. In contrast, for primary fibroblasts, ocular strains elicited significant increases in the pro- and anti-inflammatory mediators macrophage inflammatory protein (MIP)-1β, thymus- and activation-regulated chemokine (TARC), interleukin (IL)-2, IL-12p70, and interferon gamma-induced protein 10 (IP-10) compared to levels in urogenital strains, while urogenital strains elicited a distinct and significant increase in the proinflammatory mediators IL-1α, IL-1β, IL-8, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Our data indicate that primary fibroblasts, not epithelial cells, drive host inflammatory responses that are dependent on strain type and likely influence disease outcomes, establishing their importance as a novel model for studies of C. trachomatis disease pathogenesis.IMPORTANCEChlamydia trachomatis is a human pathogen and the leading cause of preventable blindness and sexually transmitted diseases in the world. Certain C. trachomatis strains cause ocular disease, while others cause upper genital tract pathology. However, little is known about the cellular or immunologic basis for these differences. Here, we compared the abilities of the strain types to infect, replicate, and initiate an immune response in primary human ocular and urogenital epithelial cells, as well as in fibroblasts from the underlying stroma. While there were no significant differences in infection rates or intracellular growth for any strain in any cell type, proinflammatory responses were driven not by the epithelial cells but by fibroblasts and were distinct between ocular and urogenital strains. Our findings suggest that primary fibroblasts are a novel and more appropriate model for studies of immune responses that will expand our understanding of the differential pathological disease outcomes caused by various C. trachomatis strain types.

Chlamydia suis is a swine pathogen that also causes zoonotic infections. Many strains contain a tet(C) cassette conferring tetracycline resistance originally acquired by horizontal gene transfer from a Gram-negative donor. As we and others have described the capacity for Chlamydiaceae to exchange DNA by homologous recombination, tet(C)-containing C. suis strains represent a potential source for antibiotic-resistance spread within and between Chlamydia species. Here, we examine the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by plaque assay. C. suis strain Rogers132, which lacks IS200/IS605 transposases, was the most efficient donor, producing three confirmed recombinants of 56 (5.4%) clones with a minimal inhibitory concentration (MIC) of ~8µg/mL. Resistance was stable when recombinants were grown initially in tetracycline at twice the MIC of S45 (0.032 µg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two sites in different recombinants: a 55kb exchange between nrqF and pckG, and a 175kb exchange between kdsA and cysQ. Neither site was associated with repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydia species that infect humans, a matter of public health importance.

Chlamydia pneumoniae is responsible for a high prevalence of respiratory infections worldwide and has been implicated in atherosclerosis. Inflammation is regulated by transcription factor (TF) networks. Yet, the core TF network triggered by chlamydiae remains largely unknown. Primary human coronary artery endothelial cells were mock-infected or infected with C. pneumoniae to generate human transcriptome data throughout the chlamydial developmental cycle. Using systems network analysis, the predominant TF network involved receptor, binding and adhesion and immune response complexes. Cells transfected with interfering RNA against activator protein-1 (AP-1) members FOS, FOSB, JUN and JUNB had significantly decreased expression and protein levels of inflammatory mediators interleukin (IL)6, IL8, CD38 and tumour necrosis factor compared with controls. These mediators have been shown to be associated with C. pneumoniae disease. Expression of AP-1 components was regulated by MAPK3K8, a MAPK pathway component. Additionally, knock-down of JUN and FOS showed significantly decreased expression of Toll-like receptor (TLR)3 during infection, implicating JUN and FOS in TLR3 regulation. TLR3 stimulation led to elevated IL8. These findings suggest that C. pneumoniae initiates signalling via TLR3 and MAPK that activate AP-1, a known immune activator in other bacteria not previously shown for chlamydiae, triggering inflammation linked to C. pneumoniae disease.

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.