The contribution of Chlamydia-specific CD8⁺ T cells to upper genital tract pathology.

Genital chlamydial infections lead to severe upper reproductive tract pathology in a subset of untreated women. We demonstrated previously that tumor necrosis factor (TNF)-α-producing CD8(+) T cells contribute significantly to chlamydial upper genital tract pathology in female mice. In addition, we observed that minimal chlamydial oviduct pathology develops in OT-1 transgenic (OT-1) mice, wherein the CD8(+) T-cell repertoire is restricted to recognition of the ovalbumin peptide Ova(257-264), suggesting that non-Chlamydia-specific CD8(+) T cells may not be responsible for chlamydial pathogenesis. In the current study, we evaluated whether antigen-specific CD8(+) T cells mediate chlamydial pathology. Groups of wild-type (WT) C57BL/6J, OT-1 mice, and OT-1 mice replete with WT CD8(+) T cells (1 × 10(6) cells per mouse intravenously) were infected intravaginally with C. muridarum (5 × 10(4) IFU/mouse). Serum total anti-Chlamydia antibody and total splenic anti-Chlamydia interferon (IFN)-γ and TNF-α responses were comparable among the three groups of animals. However, Chlamydia-specific IFN-γ and TNF-α production from purified splenic CD8(+) T cells of OT-1 mice was minimal, whereas responses in OT-1 mice replete with WT CD8(+) T cells were comparable to those in WT animals. Vaginal chlamydial clearance was comparable between the three groups of mice. Importantly, the incidence and severity of oviduct and uterine horn pathology was significantly reduced in OT-1 mice but reverted to WT levels in OT-1 mice replete with WT CD8(+) T cells. Collectively, these results demonstrate that Chlamydia-specific CD8(+) T cells contribute significantly to upper genital tract pathology.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

BACKGROUND: We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. METHODS: In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. RESULTS: TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. CONCLUSIONS: Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

Protective anti-chlamydial vaccine regimen-induced CD4+ T cell response mediates early inhibition of pathogenic CD8+ T cell response following genital challenge.

We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies.

In Vitro and In Vivo Activity of (Trifluoromethyl)pyridines as Anti-Chlamydia trachomatis Agents.

Chlamydia trachomatis is the leading pathogen in sexually transmitted bacterial infections across the globe. The development of a selective treatment against this pathogen could be an attractive therapeutic option that will reduce the overuse of broad-spectrum antibiotics. Previously, we reported some sulfonylpyridine-based compounds that showed selectivity against C. trachomatis. Here, we describe a set of related compounds that display enhanced anti-chlamydial potency when compared to our early leads. We found that the active molecules are bactericidal and have no impact on Staphylococcus aureus or Escherichia coli strains. Importantly, the molecules were not toxic to mammalian cells. Furthermore, a combination of molecule 20 (the most active molecule) and azithromycin at subinhibitory concentrations acted synergistically to inhibit chlamydial growth. Molecule 20 also eradicated Chlamydia in a 3D infection model and accelerated the recovery of Chlamydia-infected mice. This work presents compounds that could be further developed to be used alone or in combination with existing treatment regimens against chlamydial infections.

Production, characterization, and antimicrobial activity of a bacteriocin from newly isolated Enterococcus faecium IJ-31.

This work aimed to isolate and characterize Enterococcus spp. from indigenous dairy products in Islamabad, Pakistan. By classical microbiological techniques, one strain from a butter sample was identified to be Enterococcus faecium, and we designated it E. faecium IJ-31. The precise identity of this strain was then established by determining the sequence of its 16S and 23S rRNA genes. The sequence homology searches revealed matches with a number of previously reported strains, such as E. faecium HN-N3 and HN-N29, both isolated from swine intestines in China. The newly isolated strain was tested for hemolysis and antibiotic sensitivity; it was nonhemolytic on sheep and human blood and sensitive to vancomycin. Consistent with its vancomycin sensitivity, repeated attempts to amplify the vancomycin resistance genes vanA and vanB failed. Similar attempts to amplify the virulence genes gelE, agg, and cyl also failed, suggesting the absence of these genes. In contrast, the enterocin-P gene, entP, readily amplified with primers based on the previously reported sequences, and the deduced sequence showed near identity with a number of reported sequences from E. faecium. Further, the 71-residue enterocin-P sequence from strain IJ-31 is only the second complete sequence reported. The enterocin was partially purified and tested for antibacterial activity. It showed potent inhibitory activity against many bacteria, including Listeria monocytogenes, a routinely used test strain. Further, the enterocin showed potent activity against Bacillus subtilis and Bacillus cereus. The enterocin retained antibacterial activity even following heating to 121 degrees Celsius for 15 min. Further, it also retained activity after exposure to pH values ranging from 4 to 10. However, proteinase K treatment rendered the peptide nonfunctional.

Tumor necrosis factor receptor superfamily members 1a and 1b contribute to exacerbation of atherosclerosis by Chlamydia pneumoniae in mice.

The host immune responses that mediate Chlamydia-induced chronic disease sequelae are incompletely understood. The role of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2), in Chlamydia pneumoniae (CPN)-induced atherosclerosis was studied using the high-fat diet-fed male C57BL/6J mouse model. Following intranasal CPN infection, TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, and TNFR 1/2 double-knockout, displayed comparable serum anti-chlamydial antibody response, splenic antigen-specific cytokine response, and serum cholesterol profiles compared to wild type (WT) animals. However, atherosclerotic pathology in each CPN-infected KO mouse group was reduced significantly compared to WT mice, suggesting that both TNFR1 and TNFR2 promote CPN-induced atherosclerosis.

Elimination of Mycoplasma contamination in Chlamydia stocks as a result of in vivo passage or plaque isolation.

OBJECTIVE: This study aims to eliminate Mycoplasma spp. contamination from laboratory stocks of Chlamydia spp. by in vivo passage or by plaque assay. RESULTS: We have described two methods of eliminating Mycoplasma contamination from Chlamydia laboratory stocks. We conclude that Mycoplasma species commonly contaminating chlamydial stocks do not survive passage in mice. Chlamydia may also be derived Mycoplasma-free by plaque assay.

CD8+ T cells mediate Chlamydia pneumoniae-induced atherosclerosis in mice.

Chlamydia pneumoniae is a community-acquired bacterial pathogen that has been strongly associated with exacerbation of atherosclerosis. We evaluated the role of CD8(+) T cells in the C57BL/6J mouse model of C. pneumoniae-induced atherosclerosis. Groups of 4- to 6-week-old male wild-type C57BL/6J (WT) mice and mice with a gene deficiency in CD8α (CD8 KO mice) were infected with C. pneumoniae and fed a high fat (HF) diet. Serum antibody response and serum cholesterol were comparable between infected CD8 KO and WT mice. However, infected CD8 KO mice displayed significantly reduced atherosclerotic plaque lesions on day 100 compared to infected WT mice, at a level comparable to both uninfected WT and CD8 KO mice fed the HF diet. Moreover, repletion of CD8 KO mice with WT CD8(+) T cells (1 × 10(7) cells/mouse intravenously) at the time of infection reverted atherosclerotic plaque lesions to WT levels. These results demonstrate that CD8(+) T cells play an important role in mediating C. pneumoniae-induced exacerbation of atherosclerotic pathology.

OT-1 mice display minimal upper genital tract pathology following primary intravaginal Chlamydia muridarum infection.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide and leads to serious pathological sequelae in the upper genital tract (UGT) including pelvic inflammatory disease, ectopic pregnancy, and infertility. Several components of the host immune responses have been shown to contribute to the UGT pathology following genital chlamydial infection. We have shown recently that CD8(+) T cells induce the chlamydial UGT pathology via the production of TNF-α. However, those studies did not determine whether the pathology is mediated by bystander or antigen-specific CD8(+) T cells. In this study, we compared chlamydial clearance and UGT pathology in OT-1 transgenic mice and the corresponding C57BL/6J wild-type mice following primary intravaginal Chlamydia muridarum infection. All CD8(+) T cells in the OT-1 mice respond only to the Ova 257-264 peptide and are incapable of responding to other antigenic epitopes including those of Chlamydia. OT-1 mice displayed vaginal chlamydial clearance comparable to the wild-type animals. However, both oviduct and uterine horn pathology were minimal in the OT-1 mice compared with the high degree of pathology observed in the wild-type animals. These results strongly suggest that Chlamydia-specific, not bystander, CD8(+) T cells mediate the UGT pathological sequelae following genital chlamydial infection.

Intranasal vaccination with Chlamydia pneumoniae induces cross-species immunity against genital Chlamydia muridarum challenge in mice.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the world and specifically in the United States, with the highest incidence in age-groups 14-19 years. In a subset of females, the C. trachomatis genital infection leads to serious pathological sequelae including pelvic inflammatory disease, ectopic pregnancy, and infertility. Chlamydia pneumoniae, another member of the same genus, is a common cause of community acquired respiratory infection with significant number of children aged 5-14 yr displaying sero-conversion. Since these bacteriae share several antigenic determinants, we evaluated whether intranasal immunization with live C. pneumoniae (1×10(6) inclusion forming units; IFU) in 5 week old female C57BL/6 mice would induce cross-species protection against subsequent intravaginal challenge with Chlamydia muridarum (5×10(4) IFU), which causes a similar genital infection and pathology in mice as C. trachomatis in humans. Mice vaccinated intranasally with live C. pneumoniae, but not mock (PBS) immunized animals, displayed high levels of splenic cellular antigen-specific IFN-γ production and serum antibody response against C. muridarum and C. trachomatis. Mice vaccinated with C. pneumoniae displayed a significant reduction in the vaginal C. muridarum shedding as early as day 12 after secondary i.vag. challenge compared to PBS (mock) immunized mice. At day 19 after C. muridarum challenge, 100% of C. pneumoniae vaccinated mice had cleared the infection compared to none (0%) of the mock immunized mice, which cleared the infection by day 27. At day 80 after C. muridarum challenge, C. pneumoniae vaccinated mice displayed a significant reduction in the incidence (50%) and degree of hydrosalpinx compared to mock immunized animals (100%). These results suggest that respiratory C. pneumoniae infection induces accelerated chlamydial clearance and reduction of oviduct pathology following genital C. muridarum challenge, and may have important implications to the C. trachomatis-induced reproductive disease in humans.

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

BACKGROUND: Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918. METHODS AND FINDINGS: The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed. CONCLUSIONS: F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components.