In Vitro Passage Selects for Chlamydia muridarum with Enhanced Infectivity in Cultured Cells but Attenuated Pathogenicity in Mouse Upper Genital Tract

Abstract

bAlthough modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity. Infection with Chlamydia trachomatis in the lower genital tract (LGT) of women can lead to upper genital tract (UGT) inflammatory pathologies, such as hydrosalpinx, resulting in complications including ectopic pregnancy and infertility (1, 2). Hydrosalpinx, which is detectable by laparoscopic examination, has been used as a surrogate marker for tubal factor infertility in women (3, 4). However, the mechanisms by which C. trachomatis induces hydrosalpinges remain unknown. The murine pathogen Chlamydia muridarum, although not known to cause human diseases, has been extensively used for studying the mechanisms of C. trachomatis pathogenesis and immunity (5-8). This is due primarily to the ease of intravaginal infection of mice with C. muridarum organisms and their ability to induce hydrosalpinx in the oviduct, leading to mouse infertility (5, 9).Both C. trachomatis and C. muridarum share a highly conserved biphasic growth cycle, which begins with the attachment of an infectious elementary body (EB) to a host cell. Multiple putative chlamydial factors, such as the major outer membrane protein (MOMP) (10-13), outer membrane complex (OMC) protein B (OmcB) (14-16), and the polymorphic membrane proteins (Pmps) (17-19), and host-derived factors, such as heparin sulfate (16,20), epidermal growth factor receptor (EGFR) (21), estrogen receptor (22), and insulin-like growth factor 2 receptor (23), have been proposed to mediate chlamydial interactions with host cells. However, the precise structural basis of the interactions between an EB and a host cell during chlamydial infection in animals and humans remains ill defined. Following attachment to epithelia...

In VivoandEx VivoImaging Reveals a Long-Lasting Chlamydial Infection in the Mouse Gastrointestinal Tract following Genital Tract Inoculation

“…Both resulted in downstream frame shifts and premature determination codons in the gene coding for TC0412. Although different mutations in this gene from C. trachomatis were found to affect the infectivity of C. trachomatis in the mouse lower genital tract (39), mutations in this gene were shown to have no significant effect on either the infectivity or the pathogenicity of C. muridarum in the mouse genital tract (36). Both the G0.1.1 and G5-pGFP-Luci C. muridarum organisms were propagated in HeLa cells and purified as elementary bodies as mentioned above.…”

“…The immunofluorescence assays used for titrating live organisms were carried out as described previously (8,36,40). For titrating the live organisms recovered from a given sample, the mean number of inclusions per view was derived from counting five random views.…”

“…The full-genome sequence of this C. muridarum strain, designated Nigg3 or CMG0, is available under GenBank accession number CP009760.1. A clone was isolated from the Nigg3 or CMG0 stock using a plaque assay (35), and the clone designated Nigg3G0.1.1 or G0.1.1 for short (36) was used in the current study. The full genome sequence of Nigg3G0.1.1 is available under GenBank accession number CP009608.1.…”

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.…”

MicroRNAs Modulate Pathogenesis Resulting from Chlamydial Infection in Mice

“…The exact percentage representation of the variants in both Nigg A and Nigg V has not been determined, although Jasper et al have presented a summary of important single nucleotide polymorphisms (SNPs) for Nigg A (32). Chen and colleagues have shown that a change in variant representation in C. muridarum can occur through in vitro passage (33). Therefore, we used these two populations to determine if disease outcome during infection with a population of variants is associated with expression of a given miRNA profile.…”