We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface–bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp–Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp–Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH₂-terminal α-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp α-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

Etiology, transmission and protection: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection (STI) globally. However, C. trachomatis also causes trachoma in endemic areas, mostly Africa and the Middle East, and is a leading cause of preventable blindness worldwide. Epidemiology, incidence and prevalence: The World Health Organization estimates 131 million new cases of C. trachomatis genital infection occur annually. Globally, infection is most prevalent in young women and men (14-25 years), likely driven by asymptomatic infection, inadequate partner treatment and delayed development of protective immunity. Pathology/Symptomatology: C. trachomatis infects susceptible squamocolumnar or transitional epithelial cells, leading to cervicitis in women and urethritis in men. Symptoms are often mild or absent but ascending infection in some women may lead to Pelvic Inflammatory Disease (PID), resulting in reproductive sequelae such as ectopic pregnancy, infertility and chronic pelvic pain. Complications of infection in men include epididymitis and reactive arthritis. Molecular mechanisms of infection: Chlamydiae manipulate an array of host processes to support their obligate intracellular developmental cycle. This leads to activation of signaling pathways resulting in disproportionate influx of innate cells and the release of tissue damaging proteins and pro-inflammatory cytokines. Treatment and curability: Uncomplicated urogenital infection is treated with azithromycin (1 g, single dose) or doxycycline (100 mg twice daily x 7 days). However, antimicrobial treatment does not ameliorate established disease. Drug resistance is rare but treatment failures have been described. Development of an effective vaccine that protects against upper tract disease or that limits transmission remains an important goal.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.

Chlamydia trachomatis is an obligate intracellular gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis. We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.

Interleukin 17 (IL-17) contributes to development of Th1 immunity and neutrophil influx duringChlamydia muridarumpulmonary infection, but its role duringC. muridarumgenital tract infection has not been described. We detected similar numbers ofChlamydia-specific Th17 and Th1 cells in iliac nodes of wild-type mice early during genitalC. muridaruminfection, while Th1 cells predominated later.il17ra^−/−mice exhibited a reduced chlamydia-specific Th1 response in draining iliac nodes and decreased local IFN-γ production. Neutrophil influx into the genital tract was also decreased. However,il17ra^−/−mice resolved infection normally, and no difference in pathology was observed compared to the wild type. Macrophage influx and tumor necrosis factor alpha (TNF-α) production were increased inil17ra^−/−mice, providing a compensatory mechanism to effectively control chlamydial genital tract infection despite a reduced Th1 response. Inifnγ^−/−mice, a marked increase in cellular infiltrates and chronic pathology was associated with an increased Th17 response. Although neutralization of IL-17 inifnγ^−/−mice decreased neutrophil influx, macrophage infiltration remained intact and the bacterial burden was not increased. Collectively, these results indicate that IL-17 contributes to the generation of Th1 immunity and neutrophil recruitment but is not required for macrophage influx or normal resolution ofC. muridarumgenital infection. These data highlight the redundant immune mechanisms operative at this mucosal site and the importance of examining site-specific responses to mucosal pathogens.

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum , CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA , was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis , glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.