dHydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice.

The recent success in transformation of Chlamydia trachomatis represents a major advancement in Chlamydia research. Plasmid-free C. trachomatis serovar L2 organisms can be transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT. Deletion of plasmid genes coding for Pgp1 to Pgp8 in pBRCT led to the identification of Pgp1, -2, -6, and -8 as plasmid maintenance factors; Pgp4 as a transcriptional regulator of chlamydial virulence-associated gene expression; and Pgp3, -5, and -7 as being dispensable for chlamydial growth in vitro. Using the pGFP::SW2 vector system, we confirmed these findings in the current report. To further dissect the roles of pgp coding sequences and Pgp proteins in plasmid maintenance, we introduced premature stop codons into the pgp genes. Stable transformants were obtained with pGFP::SW2 derivatives carrying premature stop codons in pgp8 but not in pgp1, pgp2, and pgp6, suggesting that the pgp8 coding sequence but not the Pgp8 protein is required for maintaining the plasmid, while Pgp1, -2, and -6 proteins are necessary for plasmid maintenance. We also found that a minimum of 30 nucleotides in the pgp3 coding region was required for pgp4 expression. Finally, mCherry red fluorescent protein was successfully expressed when the mCherry gene was used to replace the pgp3, pgp4, or pgp5 coding region, indicating that these regions can be used to express nonchlamydial genes in chlamydial organisms. These novel observations have provided information for further use of chlamydial plasmid shuttle vectors as genetic tools to understand chlamydial biology and pathogenicity as well as to develop attenuated chlamydial vaccines.

c Plasmid-free Chlamydia trachomatis and Chlamydia muridarum fail to induce severe pathology. To evaluate whether the attenuated pathogenicity is due to insufficient infection or inability of the plasmidless chlamydial organisms to trigger pathological responses, we compared plasmid-competent and plasmid-free C. muridarum infections in 5 different strains of mice. All 5 strains developed hydrosalpinx following intravaginal inoculation with plasmid-competent, but not inoculation with plasmidfree, C. muridarum. The lack of hydrosalpinx induction by plasmid-free C. muridarum correlated with significantly reduced live organism recovery from the lower genital tract and shortened infection in the upper genital tract. The plasmid-free C. muridarum organisms failed to induce hydrosalpinx even when the organisms were directly inoculated into the oviduct via an intrabursal injection, which was accompanied by significantly reduced survival of the plasmidless organisms in the genital tracts. Furthermore, plasmid-competent C. muridarum organisms after UV inactivation were no longer able to induce hydrosalpinx even when directly delivered into the oviduct at a high dose. Together, these observations suggest that decreased survival of and shortened infection with plasmid-free C. muridarum may contribute significantly to its attenuated pathogenicity. We conclude that adequate live chlamydial infection in the oviduct may be necessary to induce hydrosalpinx.

Intravaginal infection with Chlamydia muridarum in mice can ascend to the upper genital tract, resulting in hydrosalpinx, a pathological hallmark for tubal infertility in women infected with C. trachomatis. Here, we utilized in vivo imaging of C. muridarum infection in mice following an intravaginal inoculation and confirmed the rapid ascent of the chlamydial organisms from the lower to upper genital tracts. Unexpectedly, the C. muridarum-derived signal was still detectable in the abdominal area 100 days after inoculation. Ex vivo imaging of the mouse organs revealed that the long-lasting presence of the chlamydial signal was restricted to the gastrointestinal (GI) tract, which was validated by directly measuring the chlamydial live organisms and genomes in the same organs. The C. muridarum organisms spreading from the genital to the GI tracts were detected in different mouse strains and appeared to be independent of oral or rectal routes. Mice prevented from orally taking up excretions also developed the long-lasting GI tract infection. Inoculation of C. muridarum directly into the upper genital tract, which resulted in a delayed vaginal shedding of live organisms, accelerated the chlamydial spreading to the GI tract. Thus, we have demonstrated that the genital tract chlamydial organisms may use a systemic route to spread to and establish a long-lasting infection in the GI tract. The significance of the chlamydial spreading from the genital to GI tracts is discussed.

The female lower genital tract is constantly exposed to microbial infection, some of which can ascend to and cause pathology such as hydrosalpinx in the upper genital tract, which can affect fertility. To understand host mechanisms for preventing upper genital tract pathology, we screened 11 inbred strains of mice for hydrosalpinx induction by C. muridarum. When examined on days 60 to 80 after intravaginal infection, the 11 strains fell into 3 groups based on their hydrosalpinx severity: CBA/J and SJL/J mice were highly susceptible with a hydrosalpinx score of 5 or greater; Balb/c, C57BL/6J, C57BL/10J, C3H/HeJ and C3H/HeN were susceptible with a score between 1 and <5; NOD/ShiLtJ, DBA/1J, DBA/2J and A/J were resistant with a score of <1. The diverse range of mouse susceptibility to hydrosalpinx induction may reflect the varied clinical outcomes of C. trachomatis-infected women. When the 11 strains were infected via an intrauterine inoculation to bypass the requirement for ascension, higher incidence and more severe hydrosalpinges were induced in most mice, indicating that the interaction between chlamydial ascension and host control of ascension is critical for determining susceptibility to hydrosalpinx development in many mice. However, a few mouse strains resisted significant exacerbation of hydrosalpinx by intrauterine infection, indicating that these mice have evolved ascension-independent mechanisms for preventing upper genital tract pathology. Together, the above observations have demonstrated that different strains of mice can prevent upper genital tract pathology by using different mechanisms.