Plasmid-Encoded Pgp3 Is a Major Virulence Factor for Chlamydia muridarum To Induce Hydrosalpinx in Mice

Abstract

Hydrosalpinx 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.

INTRODUCTION

Chlamydia trachomatis infection in the lower genital tract (LGT) of a woman can lead to inflammatory pathologies such as hydrosalpinx in the upper genital tract (UGT), resulting in complications, including ectopic pregnancy and infertility (1, 2). However, the mechanisms by which C. trachomatis organisms ascend and induce hydrosalpinges remain unknown, and it has been difficult to directly define the virulence factors of C. trachomatis. The species C. muridarum, although causing no known human diseases, has been extensively used to study the mechanisms of C. trachomatis pathogenesis and immunity (3–9). This is because intravaginal infection of mice with C. muridarum can also induce hydrosalpinx in the oviduct, leading to mouse infertility (4, 10). We have recently optimized the C. muridarum mouse model by visually detecting long-lasting hydrosalpinges at least 8 weeks after infection (9, 11–14). These C. muridarum murine model-based studies have led us to hypothesize that both adequate ascension to and induction of the appropriate inflammatory responses in the UGT are necessary for hydrosalpinx development. However, the virulence factors required for C. muridarum pathogenicity that lead to tissue inflammation and pathology have not been identified.

Almost all C. trachomatis clinical isolates contain a highly conserved plasmid coding for eight open reading frames designated Pgp1 to -8 (15–18). This plasmid may play a significant role in C. trachomatis pathogenesis, since plasmid-free (PF) C. trachomatis exhibited significantly reduced pathogenicity in ocular tissues of primates (19) and genital tract tissues of mice (20). These findings are consistent with previous observations that C. muridarum induction of hydrosalpinx in mice is plasmid dependent (8, 9). The recent success in transforming C. trachomatis (21–28) has allowed chlamydiologists to further identify and characterize the C. trachomatis plasmid-encoded pathogenic determinants. For example, the plasmid genes coding for Pgp3, -4, -5, and -7 can be deleted from C. trachomatis serovar L2 (22, 23). Pgp4 regulates the expression of many plasmid- and chromosome-borne genes and thus is considered a master regulator. In contrast, deficiencies in Pgp3, -5, or -7 did not significantly affect the expression of other chlamydial genes. Ramsey et al. (29) recently evaluated an L2 organism with a pgp3 gene deletion in the mouse model and found that the Pgp3 deficiency significantly attenuated L2 infectivity and pathogenicity in the mouse genital tract. However, because of the failure of the wild-type L2 organisms to induce hydrosalpinx in mice, it remains unclear whether Pgp3 or any other plasmid-encoded factors are required for chlamydial induction of the long-lasting hydrosalpinx (11, 30) that is most relevant to C. trachomatis-induced tubal factor infertility in women (31). We recently optimized the chlamydial transformation system for C. muridarum transformation and found that C. muridarum plasmid-encoded Pgp4 could positively regulate many plasmid-dependent genes while Pgp5 exhibited negative regulation (32). However, a deficiency in either Pgp3 or Pgp7 failed to significantly affect the expression of other chlamydial genes, which is consistent with observations in C. trachomatis L2 transformants. These single-gene-deficient C. muridarum mutants have made it possible to determine the roles of C. muridarum plasmid-borne genes in C. muridarum induction of hydrosalpinx in mice.

In the present study, we evaluated the C. muridarum transformants deficient in Pgp3, -4, or -7 for hydrosalpinx induction in mice. We found that deficiency in either Pgp3 or -4 but not -7 completely abolished the ability of C. muridarum to induce hydrosalpinx. Since Pgp4 regulates pgp3 expression but Pgp3 does not affect Pgp4 function, Pgp3 is considered a primary virulence factor. Further characterization of the Pgp3-deficient transformants has revealed important roles for Pgp3 in facilitating chlamydial survival in the mouse lower and UGT. Thus, we have identified Pgp3 as a major virulence factor in C. muridarum pathogenicity in mice.

MATERIALS AND METHODS

Chlamydial organisms and infection.

Wild-type (WT) cryptic-plasmid-containing C. muridarum (Nigg) organisms were propagated, purified, aliquoted, and stored as described previously (33). HeLa cells (human cervical epithelial carcinoma cells, catalog number CCL2) were purchased from the American Type Culture Collection (Manassas, VA). For chlamydial infection in cell culture, cells grown in 24-well plates with or without coverslips, in 6-well plates, or in flasks containing Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA) at 37°C in an incubator supplied with 5% CO₂ were inoculated with C. muridarum as described previously (33). PF C. muridarum organisms (clone CMUT3) were generated by using novobiocin (catalog no. 74675; Sigma-Aldrich, St. Louis, MO) as described previously (9, 34). Both the initial PF organisms and the subsequent transformants were plaque cloned with a standard plaque assay as described previously (32, 35).

Generation of C. muridarum transformants.

The pGFP::CM shuttle vector (32) was used as the template to generate plasmids with a pgp3, -4, or -7 deletion or a premature termination codon insertion by an in-fusion cloning technique as described previously (24). Plasmids with a pgp3, -4, or -7 gene deletion were described previously (32). To construct a pGFP::CM plasmid with premature termination in the translation of the Pgp3, -4, or -7 protein, a stop codon was introduced into the 9th residue (glutamine or Q) codon of pgp3 (the glutamine codon CAA was changed to the termination codon TAA), the 13th residue (lysine or K) codon of pgp4 (AAA to TAA), and the 17th residue (glutamic acid or E) codon of pgp7 (GAA to TAA), respectively, with a QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) by following the manufacturer's instructions. Briefly, the pGFP::CM plasmid was amplified with the appropriate primers that incorporate the desired nucleotide substitutions by PCR with AccuPrime Pfx SuperMix. PCR products were purified and digested with FastDigest DpnI to remove the template DNA. The digested PCR products were purified, recombined, and transformed into Escherichia coli XL-1 blue competent cells. Plasmids with the corresponding stop codon insertions were designated pgp3S, pgp4S, and pgp7S, respectively. These mutation-bearing plasmids were used along with the WT plasmid to transform PF C. muridarum CMUT3 as described previously (24, 32) to obtain transformants CM-pgp3S, CM-pgp4S, CM-Pgp7S, and CM-pGFP::CM, respectively. All transformants were plaque purified as described previously (35) for further experiments.

In vitro characterization of C. muridarum transformants.

An immunofluorescence assay was carried out to detect Pgp3 and GlgA, respectively, by a triple staining technique as described previously (23, 32). Briefly, HeLa cells grown on coverslips were fixed with 2% (vol/vol) paraformaldehyde (Sigma) dissolved in phosphate-buffered saline for 30 min at room temperature, followed by permeabilization with 2% (wt/vol) saponin (Sigma) for an additional 30 min. After washing and blocking, cell samples were subjected to antibody and chemical staining. Hoechst (blue; Sigma) was used to visualize DNA. A rabbit antichlamydial antibody plus a goat anti-rabbit IgG secondary antibody conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used to visualize chlamydial organism-containing inclusions. Mouse antibodies against Pgp3 or GlgA in combination with a goat anti-mouse IgG secondary antibody conjugated with Cy3 (red; Jackson ImmunoResearch Laboratories) were used to visualize the Pgp3 or GlgA protein. Immunofluorescence images were acquired with an Olympus AX-70 fluorescence microscope equipped with multiple filter sets and Simple PCI imaging software (Olympus) as described previously (36). The images were processed with the Adobe Photoshop program (Adobe Systems, San Jose, CA).

Iodine staining of accumulated glycogen was carried out as described previously (23, 24, 32). Briefly, C. muridarum-infected cultures were fixed with ice-cold methanol for 10 min and stained with 5% iodine stain (5% potassium iodide and 5% iodine in 50% ethanol) for 40 min. Individual coverslips were mounted in 50% glycerol containing 5% potassium iodide and 5% iodine. Images were acquired with an Olympus CH-30 microscope equipped with a Canon EOS Rebel T3i digital SLR camera and processed with Adobe Photoshop.

The plasmid copy number per genome was calculated on the basis of quantitative PCR detection of both plasmid and genome copy numbers in the same samples (32).

Mouse infection and live organism recovery from mouse vaginal/cervical swabs and tissue homogenates.

The WT C. muridarum Nigg strain or PF C. muridarum CMUT3 with or without transformation with the full plasmid or plasmids with gene deletion or premature termination were used to infect female C3H/HeJ mice (6 to 7 weeks old; The Jackson Laboratory, Bar Harbor, ME) intravaginally or intrabursally (37) with 2 × 10⁵ inclusion-forming units (IFU). Five days prior to infection, each mouse was injected with 2.5 mg of medroxyprogesterone (Depo-Provera; Pharmacia Upjohn, Kalamazoo, MI) subcutaneously to increase its susceptibility to infection. After infection, mice were monitored for vaginal live organism shedding and by day 60 or different days after infection (as indicated in individual experiments), mice were sacrificed to observe genital tract pathologies or determine the titers of live C. muridarum organisms in different sections of the mouse genital tract. The animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Laboratory Animal Experiments of the University of Texas Health Science Center at San Antonio.

To monitor live organism shedding from swab samples, vaginal/cervical swabs were taken every 3 to 4 days for the first week and weekly thereafter until negative shedding was observed at two consecutive time points. To quantitate live chlamydial organisms, each swab was soaked in 0.5 ml of SPG (sucrose-phosphate-glutamate buffer consisting of 218 mM sucrose, 3.76 mM KH₂PO₄, 7.1 mM K₂HPO₄, and 4.9 mM glutamate, pH 7.2) and vortexed with glass beads, and the titers of the chlamydial organisms released into the supernatants were determined on HeLa cell monolayers in duplicate (38). The infected cultures were processed for an immunofluorescence assay as described above (in the in vitro characterization section). The inclusions were counted under a fluorescence microscope. Five random fields were counted per coverslip. For coverslips with <1 IFU per field, entire coverslips were counted. Coverslips showing obvious cytotoxicity of HeLa cells were excluded. The total number of IFU per swab was calculated on the basis of the number of IFU per view, the number of views per coverslip, dilution factors, inoculation doses, and total sample volumes. An average was taken from the serially diluted and duplicate samples for any given swab. The calculated total number of IFU per swab was converted into log₁₀, and the log₁₀ IFU counts were used to calculate the mean and standard deviation at each time point.

To monitor ascending infection, mice infected intravaginally in parallel experiments were sacrificed on days 1, 3, and 10 after infection. Their entire genital tracts were sterilely harvested, and each tract was divided into three portions, including the vagina-cervix (VC), uterus-uterine horn (UH), and oviduct-ovary (OV). The VC was defined as the LGT, while both the UH and the OV were defined as the UGT. Each tissue portion was homogenized in 0.3 ml of cold SPG with a 2-ml tissue grinder (catalog number K885300-0002; Fisher Scientific, Pittsburgh, PA). After brief sonication and centrifugation at 3,000 rpm for 5 min to pellet large debris, the titers of live C. muridarum organisms in the supernatants were determined on HeLa cells as described above. The results were expressed as log₁₀ IFU counts per tissue type.

To monitor C. muridarum organism growth and spreading in the UGT, groups of mice were intrabursally inoculated with C. muridarum in the left side as described previously (9, 39). On days 1 and 7 after infection, genital tract sections with separation of the right and left sides were harvested to determine the titers of live organisms as described above, and the results were expressed as log₁₀ IFU counts per tissue type.

Genital tract pathology.

To evaluate genital tract tissue pathology, mice were sacrificed 60 days after intravaginal infection as described above. Before the removal of genital tract tissues from the mice, an in situ gross examination for evidence of hydrosalpinx formation and any other gross abnormalities was performed with a stereoscope (Olympus, Center Valley, PA). The genital tract tissues were then isolated in their entirety from the vagina to the ovary and laid on a blue sheet for acquisition of digitized images. The oviduct hydrosalpinges were visually scored on the basis of their dilation sizes by using a scoring system described previously (14). No oviduct dilation or swelling found by stereoscope inspection was defined as no hydrosalpinx and assigned a score of 0, hydrosalpinx visible only after amplification was assigned a score of 1, hydrosalpinx clearly visible to the naked eye but with a size smaller than that of the ovary was assigned a score of 2, hydrosalpinx with a size similar to that of the ovary was assigned a score of 3, and hydrosalpinx larger than the ovary was assigned a score of 4. The oviduct hydrosalpinx incidence and severity scores were analyzed for statistical significance of differences between mice infected with different C. muridarum isolates.

For histological scoring, the excised mouse genital tract tissues, after photographing, were fixed in 10% neutral formalin, embedded in paraffin, and serially sectioned longitudinally (at 5 μm/section). Efforts were made to include the cervix and both uterine horns and oviducts, as well as the lumenal structures of each tissue in each section. The sections were stained with hematoxylin and eosin (H&E) as described elsewhere (4). The H&E-stained sections were scored for severity of inflammation and pathologies on the basis of the modified schemes established previously (4, 40). Scoring for oviduct dilatation was as follows: 0, no significant dilatation; 1, mild dilatation of a single cross section; 2, one to three dilated cross sections; 3, more than three dilated cross sections; 4, confluent pronounced dilation. Inflammatory cell infiltrates were scored as follows: 0, no significant infiltration; 1, infiltration at a single focus; 2, infiltration at two to four foci; 3, infiltration at more than four foci; 4, confluent infiltration. Scores from both sides of the oviducts were added to represent the oviduct pathology of a given mouse, and the median of the individual mouse scores in each group was calculated. Thus, oviduct lumenal dilation scores and inflammation scores represent microscopic observations. Together with gross pathology parameters of hydrosalpinx incidence and severity, the combination of the four parameters allowed us to more accurately describe the oviduct pathology seen. The researchers who scored the pathology were blinded to the experimental conditions.

RESULTS

C. muridarum deficient in either Pgp3 or -4 failed to induce hydrosalpinx in mice following intravaginal infection.

Mice intravaginally inoculated with C. muridarum with or without a plasmid or PF C. muridarum transformed with or without a deficiency in plasmid-encoded proteins were compared for oviduct pathology on day 60 after infection (Fig. 1). Eighty percent of the mice infected with WT C. muridarum developed severe oviduct hydrosalpinges that were visible by the naked eye (gross pathology severity score of 4.20 ± 3.01), while none of the mice similarly infected with PF C. muridarum CMUT3 developed any detectable hydrosalpinx. When the PF C. muridarum organisms were transformed with fully competent plasmid pGFP::CM, the transformant designated CM-pGFP::CM induced severe hydrosalpinx development in 100% of the mice (gross pathology severity score of 4.78 ± 2.22). However, transformants carrying a plasmid with an in-frame deletion of either the pgp3 or the pgp4 gene (designated CM-Δpgp3 or -4) failed to induce hydrosalpinx in any mice, fully phenocopying PF C. muridarum. The transformant with a pgp7 deletion (CM-Δpgp7) induced hydrosalpinx in 57% of the mice, with a severity score of 2.29 ± 2.14. To minimize the potential polar effect of gene deletion, we further tested the transformants engineered with a premature termination codon in the pgp3, -4, or -7 gene, respectively. These transformants carry plasmids that are identical to fully competent plasmid pGFP::CM, except for one nucleotide substitution in the pgp3, -4, or -7 gene, and are designated CM-pgp3S, CM-pgp4S, or CM-pgp7S, respectively. We found that neither CM-pgp3S nor CM-pgp4S induced hydrosalpinx in any mice, while CM-pgp7S induced 75% of the mice to develop hydrosalpinx. These results confirmed the above-described findings obtained with deletion mutants. The gross pathology was further validated by microscopy (Fig. 2). We found that oviducts from mice infected with WT C. muridarum or PF CMUT3 transformed with the full plasmid pGFP::CM (CM-pGFP::CM) developed severe chronic inflammatory infiltration and lumenal dilation. However, PF CMUT3 alone or transformed with a plasmid deficient in Pgp3 (CM-Δpgp3 and CM-pgp3S) or Pgp4 (CM-Δpgp4 and CM-pgp4S) but not Pgp7 (CM-Δpgp7 and CM-pgp7S) failed to develop significant chronic inflammatory infiltration and lumenal dilation. These microscopic observations supported the gross pathology findings made by the naked eye as described above.

FIG 1.

Lack of hydrosalpinx in C3H/HeJ mice intravaginally infected with C. muridarum deficient in Pgp3 or Pgp4. WT C. muridarum (CM), PF CMUT3, or CMUT3 transformed with complete plasmid pGFP::CM (CM-pGFP::CM) or plasmids with the pgp3, -4, or -7 gene deleted (CM-Δpgp3, CM-Δpgp4, or CM-Δpgp7) or with a premature termination codon inserted into each of these genes (CM-pgp3S, CM-Pgp4S, or CM-Pgp7S) were used to intravaginally infect C3H/HeJ mice with an infective dose of 2 × 10⁵ IFU. Sixty days after infection, all of the mice were sacrificed to observe the gross appearance of the UGT. One representative image of the entire genital tract from each group is presented with the VC at the left and the OV at the right. The OV portion is magnified for identification (white arrows) and scoring (white numbers) of hydrosalpinx. The total number of mice (n) and the incidence (%) and severity (mean ± standard deviation) of hydrosalpinx in each group are shown under the corresponding images. Note that the CM (WT; panel a) and CM-pGFP::CM (c) induced a high incidence of severe hydrosalpinx, while CMUT3 alone (b) and the CM-Δpgp3 (d), CM-pgp3S (g), CM-Δpgp4 (e), and CM-pgp4S (h) transformants failed to induce any significant hydrosalpinges. &, P < 0.05; &&, P < 0.01 (Fisher's exact test); *, P < 0.05; **, P < 0.01 (Wilcoxon rank sum test).

FIG 2.

Lack of significant histopathology in C3H/HeJ mice intravaginally infected with C. muridarum deficient in Pgp3 or Pgp4. (A) The genital tract tissues harvested from mice described in the legend to Fig. 1 were subjected to histopathology examination. Microscopic images of H&E-stained sections of oviduct tissue were acquired with a 10× or 100× objective. One representative 10× image from each group is presented (left, panels a to i) and two 100× images (right, a1 to i1 and a2 to i2) taken from the areas indicated in each 10× image are also presented for each group. The severity of chronic inflammatory infiltration (B) and lumenal dilation (C) was semiquantitatively scored as described in Materials and Methods. Note that oviducts from mice infected with WT C. muridarum (CM, open circle) or PF CMUT3 (inverted open triangles) transformed with full plasmid pGFP::CM (pentagon) or plasmids deficient in Pgp3 (Δpgp3 and pgp3S; open or filled squares) but not Pgp4 (Δpgp4 and pgp4S; open or filled triangles) or Pgp7 (Δpgp7 and pgp7S; open or filled diamonds) displayed severe chronic inflammatory infiltration and lumenal dilation. **, P < 0.01 (Wilcoxon rank sum test; all compared with the full-plasmid-complemented CMUT3 group, CM-pGFP::CM).

Pgp3 is an effector virulence factor.

It has been previously shown that during C. trachomatis serovar L2 infection, Pgp4 regulates the expression of pgp3 and many other genes while Pgp3 does not significantly affect the expression of other chlamydial genes, including pgp4 (22, 23). We recently found that, similar to C. trachomatis, C. muridarum with a pgp4 deletion displayed a significant reduction in the expression of pgp3 while a deficiency in Pgp3 failed to significantly affect the expression of pgp4 (32). Since Pgp4 but not Pgp3 is known to positively regulate glgA expression and glycogen synthesis (22, 23, 32), we compared the expression of the GlgA protein and accumulation of glycogen in C. muridarum transformants with a premature termination codon inserted into pgp3, -4, or -7, respectively (Fig. 3). As expected, in cell cultures infected with CM-pgp4S, the GlgA protein was not detectable and there was no significant glycogen accumulation, confirming the roles of Pgp4 in the regulation of GlgA expression and glycogen synthesis. It is worth noting that the Pgp3 protein was not detected in the CM-pgp4S-infected culture, validating the dependence of Pgp3 protein expression on Pgp4. On the contrary, in the cultures infected with CM-pgp3S or CM-pgp7S, both GlgA expression and glycogen accumulation were obvious, indicating that lack of Pgp3 did not significantly affect the functionality of Pgp4. The plasmid copy numbers of the various transformants were also compared (Fig. 4). We observed that all transformants either with individual pgp genes deleted or with premature termination codons inserted into the corresponding pgp genes maintained similar plasmid copy numbers per genome, which were 2- to 3-fold higher than that of WT C. muridarum. These observations suggest that the plasmid copy number did not contribute to the varied gene or protein expression levels among the transformants.

FIG 3.

Effects of Pgp4 deficiency on Pgp3 and GlgA protein expression and glycogen synthesis. HeLa cells were infected with six categories of C. muridarum organisms as described the legend to Fig. 2 and shown at the top. Twenty-four hours after infection, the cells were used for triple immunofluorescence labeling for the Pgp3 (red, panels a to f) and GlgA (red, panels g to l) proteins, chlamydial organisms (green, panels a to l), DNA (blue, panels a to l), and iodine staining of glycogen accumulation (panels m to r). Note that both GlgA expression and glycogen accumulation were restored in cultures infected with C. muridarum deficient in pgp3 (pgp3S) or pgp7 (pgp7S) but not pgp4 (pgp4S). The Pgp3 protein was also significantly reduced in the culture infected with pgp4-deficient organisms (pgp4S). Red arrows point to either Pgp3 or GlgA, while yellow arrows point to glycogen.

FIG 4.

Plasmid copy numbers per genome in different CMUT3 transformants. HeLa cell cultures infected with the WT, PF CMUT3, or various CMUT3 transformants as shown on the x axis were harvested at 20 h postinfection for quantitation of genome (16S rRNA gene) and plasmid (pgp8 gene) copy numbers by real-time PCR. The ratio of the plasmid copy number per genome to the WT plasmid copy number per genome is shown on the y axis. Note that although all of the transformants displayed significantly higher plasmid copy numbers per genome than WT C. muridarum (CM), there was no significant difference among the plasmid copy numbers of the seven transformants.

Pgp3 is required for C. muridarum survival in the mouse LGT.

We further investigated why Pgp3-deficient C. muridarum failed to induce hydrosalpinx by comparing the infection courses of mice infected with different versions of C. muridarum or C. muridarum transformants (Fig. 5). We found that mice infected with PF C. muridarum CMUT3 developed significantly lower levels of live organism shedding from the LGT on days 3, 7, and 14 after infection than mice infected with WT C. muridarum. Transformation of CMUT3 with complete plasmid pGFP::CM restored their live organism shedding to the same level as WT C. muridarum. Nevertheless, the length of live organism shedding was not significantly different between mice infected with PF or WT C. muridarum, which is consistent with a previous report (9). Importantly, the transformants deficient in either Pgp3 or -4 but not -7, whether via in-frame deletion of or premature termination codon insertion into the corresponding pgp genes, displayed significantly reduced levels of live organism shedding from their LGTs on days 3, 7, and 14 after infection. This observation has demonstrated a critical role for Pgp3 in C. muridarum infectivity in the mouse LGT. Furthermore, the similar shedding profiles of PF CMUT3 and Pgp3-deficient organisms (CM-Δpgp3 or CM-pgp3S) suggest that Pgp3 deficiency can fully phenocopy plasmid deficiency during infection of the mouse LGT.

FIG 5.

Effect of Pgp3 deficiency on live C. muridarum organism shedding from the mouse LGT. C3H/HeJ mice intravaginally infected with WT (a) or PF (b) C. muridarum and seven transformants (c to i) as described the legend to Fig. 1 were monitored for live organism shedding from the LGT (detected in vaginal swabs). The number of live C. muridarum organisms recovered is expressed as IFU counts as shown on the y axis on a log₁₀ scale. On different days postinfection, as shown on the x axis, vaginal swabs were taken to determine the titers of live organisms. Note that mice infected with PF CMUT3 or CMUT3 transformed with a plasmid either depleted of or with a premature termination codon inserted into pgp3 (CM-Δpgp3 or CM-pgp3S; d or g,) or pgp4 (CM-Δpgp4 or CM-pgp4S; e or h) but not pgp7 (CM-Δpgp7 or CM-pgp7S; f or i) displayed significantly reduced live organism shedding. *, P < 0.05; **, P < 0.01 (Krystal-Wallis test).

Pgp3 is required for C. muridarum ascension to the mouse UGT.

We further monitored live organism recovery from different sections of the genital tract to assess the role of Pgp3 in C. muridarum ascension to the UGT (Fig. 6). On day 1 after intravaginal infection, there was no significant difference in the number of live organisms recovered from the LGT, the VC, or the UGT UH tissue homogenates, regardless of the types of C. muridarum used to infect mice. However, by day 3, as the number of live C. muridarum organisms recovered from genital tract tissue homogenates of all of the mice increased, mice infected with CM-pgp3S but not CM-pgp7S displayed significantly lower levels of live organisms in both VC and UH homogenates than mice infected with CM-pGFP::CM. The same trend continued to day 10 after infection. Importantly, when the live C. muridarum organisms recovered from the oviduct-ovary (OV) homogenates were compared on day 10, PF CMUT3 displayed a significantly lower level than full-plasmid-transformed CM-pGFP::CM, confirming an essential role for the plasmid in the ascension of C. muridarum to the oviduct (9). More importantly, mice infected with CM-pgp3S but not CM-pgp7S also exhibited a significantly reduced level of live organisms from the OV homogenate, suggesting that Pgp3 is required for efficient ascension of C. muridarum to the oviduct, in which C. muridarum-induced hydrosalpinx takes place.

FIG 6.

Effect of Pgp3 deficiency on the ascension of chlamydiae to the UGT following intravaginal infection. C3H/HeJ mice intravaginally infected with PF C. muridarum CMUT3 [CMUT3(PF); open bar] or CMUT3 transformed with full plasmid pGFP::CM (CM-pGFP::CM; diagonal lines) or plasmids with a premature stop codon in the pgp3 (CM-pgp3S; horizontal lines) or pgp7 (CM-pgp7S; vertical lines) gene were sacrificed on day 1 (a), 3 (b), or 10 (c) after infection. The entire genital tract tissue was harvested from each mouse and divided into the LGT VC and the UGT UH and OV sections. Each tissue section was homogenized for determination of the titers of live C. muridarum organisms (IFU counts). The log₁₀ IFU counts were used to calculate the mean value and standard deviation of each group, as sown on the y axis. Note that the number of live organisms recovered from mice infected with CM-pgp3S but not from mice infected with CM-pgp7S was significantly lower than that from mice infected with full-plasmid-transformed CM-pGFP::CM. n = 5 per group. *, P < 0.05; **, P < 0.01 (Kruskal-Wallis analysis).

Pgp3 is required for adequate replication and spreading of C. muridarum in the mouse UGT.

Since sufficient numbers of live C. muridarum organisms in the oviduct are required for hydrosalpinx induction (9, 12), we next tested whether Pgp3 is essential for C. muridarum to infect mouse oviduct tissue. The various versions of C. muridarum organisms described in Materials and Methods and the legend to Fig. 1 were directly inoculated into the mouse oviduct via intrabursal injection on the left side, and then live organism recovery from different sections of the genital tract was monitored (Fig. 7). On day 1 after intrabursal inoculation, there were no C. muridarum organisms spreading to other parts of the genital tract, and significant amounts of live organisms were recovered only from the left uterine horn (L.UH) and the left OV (L.OV, which is the initial injection site) tissue homogenates. It is worth noting that the levels of live organisms recovered from mice infected with PF CMUT3 were significantly lower than those from mice infected with full-plasmid-transformed CM-pGFP::CM organisms, which is consistent with what we have previously reported (9). This observation suggests that the plasmid is required for C. muridarum survival in the oviduct epithelial tissue. Interestingly, mice infected with CM-pgp3S developed significant oviduct infection similar to that of CM-pGFP::CM-infected mice, suggesting that Pgp3-independent plasmid factors can promote C. muridarum survival in the UGT soon after intrabursal injection. By day 7, significant levels of live organisms were recovered from all sections of the genital tract tissues of mice infected with plasmid-competent CM-pGFP::CM, suggesting that C. muridarum organisms are able to establish a productive infection in the left oviduct and spread to the rest of the genital tract, including descending to the LGT and crossing into the right side of the genital tract. However, no increase in live organism recovery from any sections of the genital tract tissues was detected in PF CMUT3-infected mice. These observations suggest that the plasmid is necessary for optimal infection of oviduct epithelial cells by C. muridarum and efficient spreading to the rest of the genital tract, which we have reported previously (9). Importantly, the live organism recoveries from CM-pgp3S-infected mice but not CM-pgp7S-infected mice were significantly lower than those from CM-pGFP::CM-infected mice, suggesting that Pgp3 but not Pgp7 is required for the optimal growth and spreading of C. muridarum in UGT tissue. Nevertheless, comparison of CM-pgp3S with PF CMUT3 indicates that CM-pgp3S actively infected the oviduct tissues and underwent significant spreading, including descending to the LGT and migrating to the right side of the UGT. It appears that Pgp3-deficient C. muridarum (CM-pgp3S) was less attenuated than PF CMUT3 in the UGT, suggesting that Pgp3-independent plasmid factors may also contribute to C. muridarum growth and replication in the oviduct. However, Pgp3-independent C. muridarum growth and replication in the oviduct appeared to be insufficient for C. muridarum to induce hydrosalpinx.

FIG 7.

Effect of Pgp3 deficiency on chlamydial survival in the UGT. C3H/HeJ mice were infected via intrabursal injection on the left side (as indicated by the red arrow at the top) with PF C. muridarum CMUT3 (a and e) or CMUT3 transformed with full plasmid pGFP::CM (b and f) or a plasmid with a premature termination codon inserted into the pgp3 (pgp3S, c and g) or pgp7 (pgp7S, d and h) gene were sacrificed on day 1 (a to d) or 7 (e to h) after infection. The genital tract tissue was harvested from each mouse and divided into the VC, uterus, L.UH, right uterine horn (R.UH), L.OV, and right OV (R.OV) sections. Each tissue section was homogenized for determination of the titers of live C. muridarum organisms (IFU counts). The log₁₀ IFU counts were used to calculate the mean value and standard deviation for each group, as shown on the y axis. Note that the number of live organisms recovered from mice infected with CM-pgp3S but not CM-pgp7S was significantly lower than that recovered from mice infected with full-plasmid-transformed CM-pGFP::CM. n = 5 per group. *, P < 0.05; **, P < 0.01 (Kruskal-Wallis analysis).

DISCUSSION

For the first time, we have presented direct experimental evidence demonstrating a critical role for Pgp3 in C. muridarum induction of hydrosalpinx. First, C. muridarum with deletion of the pgp3 but not the pgp7 gene failed to induce any significant hydrosalpinx in the mice tested. The visual observation of hydrosalpinx was validated by microscopy. Second, the above deletion mutation results were reproduced with C. muridarum engineered with a premature termination codon in the pgp3 or pgp7 gene, which greatly minimizes potential polar effects as a result of the gene deletion. These observations have allowed us to conclude that Pgp3 plays a critical role in C. muridarum induction of hydrosalpinx. Third, although deficiency in either Pgp3 or Pgp4 resulted in the failure of C. muridarum to induce hydrosalpinx, Pgp4 is an upstream regulator while Pgp3 is likely an effector virulence molecule. This is consistent with the observation that lack of Pgp4 decreased Pgp3 expression while deficiency in Pgp3 did not affect the function of Pgp4. Fourth, deficiency in Pgp3 but not Pgp7 resulted in reduced live organism shedding from the LGT and decreased live organism recovery from the UGT after intravaginal or intrabursal infection. These experimental data together have demonstrated that Pgp3 is an essential virulence factor for C. muridarum pathogenesis in the mouse UGT.

The conclusion that Pgp3 is a virulence factor in C. muridarum induction of hydrosalpinx is also supported by previous studies. First, of the eight plasmid-encoded chlamydial proteins (16, 41), Pgp3 is the only one that is secreted into the host cell cytosol during chlamydial infection (16). Since chlamydiae can complete their biosynthesis inside the inclusion, any chlamydial proteins that are secreted outside the inclusion may be used by chlamydial organisms as virulence factors for interaction with the infected host. Second, Pgp3 is an immunodominant antigen in women who have acquired chlamydial infection via either the genital tract (42, 43) or the ocular route (38, 44). In general, chlamydia-secreted proteins are likely to be more immunogenic (43). Thus, the immunodominance of Pgp3 is consistent with its secretion into host cells. Third, Pgp3 forms a stable trimer (45) with a C-terminal trimerization domain similar to the receptor binding domain of tumor necrosis factor alpha (TNF-α) (46), which suggests that Pgp3 is poised to interact with host inflammatory responses. Finally, Pgp3-deficient C. trachomatis serovar L2 is attenuated in infecting the mouse genital tract and inducing inflammatory responses (29).

We have also presented evidence demonstrating a critical role for Pgp3 in promoting C. muridarum survival in both the LGT (Fig. 5 and 6) and the UGT (Fig. 7). Careful analyses of live organism recovery profiles revealed that the impact of Pgp3 deficiency on C. muridarum survival was detected sooner in the LGT than in the UGT. In the LGT, the Pgp3 deficiency fully phenocopied the plasmid deficiency in terms of live organism recovery on both days 1 and 3 after infection (Fig. 6), suggesting that Pgp3 or Pgp3-dependent plasmid factors played an immediate role in promoting C. muridarum infection in the LGT and the role of any potential Pgp3-independent plasmid factors was not obvious, if there was any. In the UGT, the impact of Pgp3 deficiency on C. muridarum survival was not obvious on day 1 and only became significant on day 7 during intrabursal infection (Fig. 7). The fact that Pgp3 deficiency was insufficient for phenocopying of the plasmid deficiency on both days 1 and 7 after intrabursal infection (Fig. 7) suggests that the impact of Pgp3 or Pgp3-dependent plasmid factors on C. muridarum survival in the UGT was delayed and Pgp3-independent plasmid factors might play significant roles. We are aware that these differential temporal impacts of Pgp3 deficiency on C. muridarum survival in the LGT versus the UGT still need to be confirmed by additional experiments. Nevertheless, we can now hypothesize that the immediate role of Pgp3 in the LGT may reflect Pgp3's ability to potentially mediate chlamydial attachment and promote chlamydial entry into host cells in the LGT. This hypothesis is consistent with the notion that Pgp3 is associated with the outer membrane complex (45). More likely, Pgp3 may be able to promote C. muridarum survival by neutralizing host innate defense mechanisms in the mouse genital tract since Pgp3 is both an outer membrane-associated (45) and a secreted (16) protein. The innate immunity effectors targeted by Pgp3 may preexist in the LGT (because of constitutive expression in response to the nonsterile environment of the LGT) but can only be produced in the UGT after induction by a pathogenic chlamydial infection. This hypothesis is consistent with the fact that the LGT is normally nonsterile while the UGT is sterile. The differential expression patterns of the host effectors targeted by Pgp3 in the LGT versus the UGT may partially explain the different temporal impacts of Pgp3 deficiency on C. muridarum survival in the LGT versus the UGT.

We have recently optimized the C. muridarum mouse infection model for investigation of chlamydial pathogenic mechanisms by comparing 11 inbred strains of mice for the development long-lasting hydrosalpinx in response to C. muridarum infection (12). We found that mice varied considerably in susceptibility to hydrosalpinx induction following intravaginal infection. However, most mice consistently developed more severe hydrosalpinx when they were infected with the 11 strains via intrauterine inoculation to bypass the requirement for ascension, indicating that the interaction between chlamydial ascension and host control of ascension is critical in determining susceptibility to hydrosalpinx development in many mice. In the present study, we found that Pgp3-deficent C. muridarum displayed a significantly reduced ascending infection, suggesting that Pgp3 is important for C. muridarum to overcome host control of ascension. The question is how Pgp3 promotes chlamydial ascending infection, which is under investigation. Interestingly, a few mouse strains still resisted significant exacerbation of hydrosalpinx by intrauterine infection, indicating that these mice have evolved ascension-independent mechanisms for the prevention of UGT pathology. Notably, A/J mice resisted hydrosalpinx after intrauterine infection (12). A/J mice are known to be deficient in complement factor C5, and we have found that C5 plays a critical role in C. muridarum induction of hydrosalpinx (30). Thus, host responsiveness to chlamydial infection also plays a critical role in chlamydial pathogenesis. We recently found that TNF receptor 1 (TNFR1) but not Toll-like receptor 2 played a critical role in chlamydial induction of hydrosalpinx (13), which is consistent with the observations that mice deficient in MyD88 (40, 47) still developed hydrosalpinx. The question is whether Pgp3 plays any role in C5 or TNFR1 signaling pathway activation. Since the C terminus of Pgp3 displays a trimeric fold similar to the trimer structure shared by members of the TNF-α superfamily, we hypothesize that Pgp3 may be able to activate the TNFR1 signaling pathway to promote C. muridarum induction of hydrosalpinx.