The Chromosome-Encoded Hypothetical Protein TC0668 Is an Upper Genital Tract Pathogenicity Factor of Chlamydia muridarum

Abstract

cWe previously associated a missense mutation of the tc0668 gene of serial in vitro-passaged Chlamydia muridarum, a murine model of human urogenital C. trachomatis, with severely attenuated disease development in the upper genital tract of female mice. Since these mutants also contained a TC0237 Q117E missense mutation that enhances their in vitro infectivity, an effort was made here to isolate and characterize a tc0668 single mutant to determine its individual contribution to urogenital pathogenicity. Detailed genetic analysis of C. muridarum passages revealed a truncated variant with a G216* nonsense mutation of the 408-amino-acid TC0668 protein that does not produce a detectable product. Intracellular growth and infectivity of C. muridarum in vitro remain unaffected in the absence of TC0668. Intravaginal inoculation of the TC0668 null mutant into C3H/HeJ mice results in a typical course of lower genital tract infection but, unlike a pathogenic isogenic control, is unable to elicit significant chronic inflammation of the oviduct and fails to induce hydrosalpinx. Thus, TC0668 is demonstrated as an important chromosome-encoded urogenital pathogenicity factor of C. muridarum and the first with these characteristics to be discovered for a Chlamydia pathogen. Chlamydia muridarum is a Gram-negative obligate intracellular pathogen that was isolated from a steady-state respiratory infection of laboratory mice in the early 1940s (1, 2). Like other chlamydial organisms, C. muridarum has a biphasic life cycle that alternates between infectious elementary body (EB) and replicating reticulate body (RB) morphologies. The genome of C. muridarum is reductively evolved, containing a 1.07-Mb circular chromosome and single 7.5-kb extrachromosomal plasmid (3). In that C. muridarum and other chlamydial pathogens have fewer than 1,000 genes and ϳ900 encoded proteins, roughly half the number encoded by environmental chlamydial organisms that parasitize simple single-celled eukaryotes (4), it is not known which of the many cryptic genetic factors allow them to thrive within and harm complex vertebrates.In the laboratory, C. muridarum is used as a model of urogenital disease resulting from sexually transmitted Chlamydia trachomatis in women. The basic biology and genomes of these two pathogens are highly conserved. The urogenital biovar of C. trachomatis is responsible for the most reported cases of bacterial infection in the United States (5) and is a pervasive global health problem (6). In women, ascending infection from the lower to upper genital tracts, separated by the cervical barrier, can lead to loss of the ciliated epithelium and irreversible fibrotic remodeling of the fallopian tubes after primary infection is resolved (7). If left untreated, often because of asymptomatic infection (8), afflicted women can experience severe chronic sequelae, such as tubal blockage, hydrosalpinx, spontaneous abortion, ectopic pregnancy, and tubal factor infertility (9, 10). Genital inoculation of female mice with C. muridarum results in analo...

Chlamydia muridarum with Mutations in Chromosomal Genes tc0237 and/or tc0668 Is Deficient in Colonizing the Mouse Gastrointestinal Tract

“…2). We found that both wild-type and mutant C. muridarum displayed similar courses of live-organism shedding from the lower genital tract regardless of their genotypes, confirming previous findings (15). However, all of our C. muridarum mutants…”

“…muridarum mutants that fail to induce hydrosalpinx are defective in spreading to the GI tract following intravaginal inoculation. We previously reported that a series of C. muridarum mutants were highly attenuated in induction of hydrosalpinx in the mouse upper genital tract (14,15). However, the mechanisms of attenuation remained unknown.…”

“…Thus, there may be multiple pathways/mechanisms for GI tract chlamydiae to potentially promote pathogenicity in the upper genital tract. We previously demonstrated that C. muridarum clones carrying mutations in the chromosomal genes tc0237 and/or tc0668 were highly attenuated in the induction of hydrosalpinx in the mouse upper genital tract (14,15). However, these C. muridarum mutants maintained similar live-organism shedding courses in the lower genital tract.…”

“…Chlamydia muridarum infection of the mouse genital tract can cause hydrosalpinx and infertility (4)(5)(6), which has been used to investigate the mechanisms of C. trachomatis pathogenesis (7)(8)(9)(10)(11)(12). Murine-model-based investigations have led to the conclusion that both chlamydial ascension to the upper genital tract (13)(14)(15)(16)(17) and tubal inflammation (7,11,(18)(19)(20)(21)(22)(23)(24) may be required for chlamydial induction of hydrosalpinx. However, C. muridarum infections of the genital tracts of female mice are self-limited (cleared from the entire genital tract within 4 to 6 weeks) while the resultant upper genital tract pathologies such as tubal fibrosis and hydrosalpinx can be long lasting (when examined 8 to 10 weeks later) (6,16,25,26).…”

Intravenous Inoculation with Chlamydia muridarum Leads to a Long-Lasting Infection Restricted to the Gastrointestinal Tract

“…This is because a single inoculation of C. muridarum organisms in the mouse lower genital tract can cause hydrosalpinx and infertility (7)(8)(9), closely mimicking the tubal adhesion, hydrosalpinx, and infertility observed in women urogenitally infected with C. trachomatis (10)(11)(12). Although C. muridarum is not a natural sexually transmitted agent of mice, chlamydiologists have used this model not only to identify both chlamydial (13)(14)(15)(16)(17) and host (1,5,(18)(19)(20)(21)(22)(23)(24) pathogenic determinants but also to define the roles of ascending infection and tubal inflammation in chlamydial induction of hydrosalpinx (9,16,25,26). Nevertheless, questions such as how a self-limited infection with C. muridarum in the mouse genital tract can trigger tubal fibrosis or hydrosalpinx that lasts long after the tubal infection is resolved (9,25) remain unanswered.…”