The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Although the concept of persistence in chlamydial infections has been recognized for about 80 years, there is still very little known about the mechanism by which this occurs. In this review, we revisit an old paradigm, long known to chlamydiologists and veterinarians, that in virtually all hosts of chlamydiae, including mammals and birds, chlamydiae reside in the gastrointestinal tract for long periods of time in the absence of clinical disease. Thus, if gastrointestinal infection occurs in most hosts, then it is very likely that gastrointestinal infection occurs in humans as well. We demonstrate that gastrointestinal infection does indeed occur in humans and propose that this anatomical site is the source of persistent infection in humans. The data in ruminants and animal models demonstrate that the immune system is unable to clear chlamydiae from the gut, so they can remain indefinitely, with continual shedding in feces. Clearly, many women become reinfected from an untreated partner; however, we propose that women, cured of genital infection, remain at risk for autoinoculation from the lower gastrointestinal tract. Moreover, there are substantial data demonstrating treatment failure of chlamydial infections, particularly with azithromycin. New data in the mouse model have shown that azithromycin is far less effective against chlamydial gastrointestinal infection than against genital infections. Therefore, it is possible that women cured of genital infection by antibiotics remain infected in the gastrointestinal tract and can become reinfected by autoinoculation from that site.

The mechanism by which chlamydiae persist in vivo remains undefined; however, chlamydiae in most animals persist in the gastrointestinal tract (GI) and are transmitted via the fecal-oral route. Oral infection of mice with Chlamydia muridarum was previously shown to establish a long-term persistent infection in the GI tract. In this study, BALB/c, DBA/2 and C57Bl/6 mice, infected orally with C. muridarum, were infected in the cecum for as long as 100 days in the absence of pathology. The primary target tissue was the cecum although the large intestine was also infected in most animals. A strong serum IgG and cecal IgA antibody response developed. Lymphocyte proliferation assays to chlamydial antigen on mesenteric lymph node cells were positive by day 10 and peaked on days 15–21, but the response returned to baseline levels by 50 days, despite the ongoing presence of the organism in the cecum. Since studies have shown that women and men become infected orally with chlamydiae, we propose that the GI tract is a site of persistent infection and that immune down-regulation in the gut allows chlamydiae to persist indefinitely. As a result, women may become reinfected via contamination of the genital tract from the lower GI tract.

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, aiti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, ppleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pnecvumonitis agent (MoPn), a Chiamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.

In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance of Chlamydia trachomatis from the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent of C. trachomatis (MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-␣) and interleukin-1␤ (IL-1␤) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-␣ and IL-1␤ are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.

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IFN-γ-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-β induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-β was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-β during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-β. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-β and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-β function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.

Recent findings have implicated interleukin-1␤ (IL-1␤