Gastrointestinal Coinfection Promotes Chlamydial Pathogenicity in the Genital Tract

Abstract

Sexually transmitted Chlamydia, which can cause fibrotic pathology in women's genital tracts, is also frequently detected in the gastrointestinal tract. However, the medical significance of the gastrointestinal Chlamydia remains unclear. A murine Chlamydia readily spreads from the mouse genital tract to the gastrointestinal tract while inducing oviduct fibrotic blockage or hydrosalpinx. We previously proposed a two-hit model in which the mouse gastrointestinal Chlamydia might induce the second hit to promote genital tract pathology, and we are now providing experimental evidence for testing the hypothesis. First, chlamydial mutants that are attenuated in inducing hydrosalpinx in the genital tract also reduce their colonization in the gastrointestinal tract, leading to a better correlation of chlamydial induction of hydrosalpinx with chlamydial colonization in the gastrointestinal tract than in the genital tract. Second, intragastric coinoculation with a wild-type Chlamydia rescued an attenuated Chlamydia mutant to induce hydrosalpinx, while the chlamydial mutant infection in the genital tract alone was unable to induce any significant hydrosalpinx. Finally, the coinoculated gastrointestinal Chlamydia failed to directly spread to the genital tract lumen, suggesting that gastrointestinal Chlamydia may promote genital pathology via an indirect mechanism. Thus, we have demonstrated a significant role of gastrointestinal Chlamydia in promoting pathology in the genital tract possibly via an indirect mechanism. This study provides a novel direction/dimension for further investigating chlamydial pathogenic mechanisms.

Keywords: Chlamydia; gastrointestinal colonization; genital pathology; gut chlamydia; hydrosalpinx; pathogenesis; tubal fibrosis.

FIG 1

Monitoring live organism shedding from mouse genital and GI tracts after intravaginal inoculation with chlamydial organisms with or without mutations. Groups of CBA/J mice (n = 5) intravaginally inoculated with plasmid-free (Pf) C. muridarum transformed without (CMUT3.5, panels c and d) or with pCM plasmid coding for mCherry (CMpmCherry, panels a and b, as wild-type [Wt]) or pCM with a premature stop codon installed in the pgp3 gene (CMpGP3S, panels e and f) or C. muridarum clone G13.32.1 (panels g and h) or its isogenic clones with a single chromosomal gene mutation (clone G13.11.1, panels i and j) or double gene mutation (clone 28.51.1, panels k and l) were monitored for live organism shedding from both genital (panels a, c, e, g, i, and k) and gastrointestinal (GI; panels b, d, f, h, j, and l) tracts in log₁₀ IFU (y axis) on days 3 and 7 and weekly thereafter (x axis). *, P < 0.05 (area under the curve comparison between panels b versus [v.s] panel d or f or between panel h versus panel j or i using the Wilcoxon rank sum test).

FIG 2

Detecting gross pathology from mouse genital tract following intravaginal inoculation with chlamydial organisms with or without mutations. Groups of CBA/J mice (n = 5) intravaginally inoculated with different C. muridarum organisms as described in the Fig. 1 legend (panel a for mice infected with CMpmCherry, panel b for Pf, CMUT3.5, panel c for CMpGP3S, panel d for C. muridarum wild-type clone G13.32.1, panel e for mutant clone G13.11.1, and panel f for mutant clone 28.51.1). On day 56 after infection, all mice were sacrificed for observing hydrosalpinx pathology, as described in Materials and Methods. One representative genital tract image from each group is shown with the vagina on the left and oviduct/ovary on the right. Oviducts with hydrosalpinges are indicated by white arrows. The oviduct/ovary from each side is magnified in the panels on the right, with the corresponding hydrosalpinx scores marked. The hydrosalpinx was counted and semiquantitatively graded to calculate the incidence rate and severity score in a given group.

FIG 3

Correlating mouse gross pathology hydrosalpinx with live organism recoveries from mouse genital or GI tracts. For Pearson correlation analyses, the hydrosalpinx score from each CBA1/J mouse (as displayed along the x axis) was plotted against live organism shedding in log₁₀ total numbers of IFU from genital (a) or GI (b) tract swab specimens collected from a mouse over time (y axis). Each circle represents a mouse. The total number of IFU from each mouse was obtained by adding the numbers of IFU from all time points observed. The correlation coefficient r between the hydrosalpinx scores and the number of genital or vaginal IFU was 0.5065, while that between the hydrosalpinx scores and the number of GI tract or rectal IFU was 0.6553, given that r is the square root of coefficient of determination R² in linear regression.

FIG 4

Monitoring live organism shedding from mouse genital and GI tracts after coinfection. As indicated on the left of the figure, groups of female CBA/1J mice were intravaginally (ivag) infected with a mCherry-expressing wild-type (clone CMpmCHerry, red bar [panel a, n = 10]) or plasmid-free (clone CMUT3.5, black bar [panel c, n = 14; panel e, n = 14]) chlamydial organisms. Seven days after the intravaginal infection, mice were intragastrically (ig) infected with the same Wt chlamydial organisms, as indicated by red arrows (panel f, n = 14; panel h, n = 11). All mice were monitored for live chlamydial organism shedding from both vaginal (panels a, c, e, and g) and rectal (panels b, d, f, and h) swabs, and the titers of Wt (red) and Pf (black) organisms were quantitated separately and are expressed as the log₁₀ IFU per swab (y axis) on different days after intravaginal infection (x axis). *, P < 0.01 (area-under-the-curve comparison using a Wilcoxon rank sum test).

FIG 5

Detecting gross pathology from mouse genital tract after coinfection. Groups of CBA/J mice (n = 10 to 14 per group) with or without coinfection as described in the Fig. 4 legend (panel a for mice infected intravaginally [ivag] with Wt CMpmCherry alone, panel b for ivag Pf alone, panel c for both ivag Pf and intragastric infection [ig] with Wt, and panel d for ig Wt alone). On day 56, all mice were sacrificed to determine the genital pathology macroscopically for hydrosalpinx. Only one genital tract gross image is presented as representative for the corresponding group, with the vagina on the left and the oviduct/ovary on the right (panels a to d). The hydrosalpinx (dilated oviduct accumulated with serous fluid) is marked by a white arrow. The oviduct portion is magnified for viewing the hydrosalpinx. Hydrosalpinx scores are indicated in white. Both hydrosalpinx incidence and severity score are listed on the right of the corresponding group images. *, P < 0.05 (the Wilcoxon test was used for comparing hydrosalpinx scores, while the Fisher exact test was used for comparing incidence). The data were acquired from three independent experiments.

FIG 6

Detecting oviduct dilation under microscopy after coinfection. Groups of CBA/J mice (n = 10 to 14 per group) with or without coinfection as described in the Fig. 4 legend (panel a or group 1 for mice infected ivag with Wt CMpmCherry alone, panel b or group 2 for ivag Pf alone, panel c or group 3 for both ivag Pf and ig infection with Wt, and panel d or group 4 for ig Wt alone). On day 56, all mice were sacrificed for observing microscopically for oviduct dilation. Only one microscopic image (taken under a 4× lens objective) of the genital tract tissue is presented as a representative for a given group (panels a to d). Dilated oviducts (DO) are marked by double-headed arrows, while normal oviduct (NO) are marked by single-headed arrows. The ovary is also indicated. The oviduct dilation scores from all 4 groups are displayed in panel e. Open circles are used for mouse dilation scores in groups 1 and 2, while open upright triangles are used for group 3 and upside down triangles are used for group 4.

FIG 7

Monitoring live organism shedding from mice coinoculated with both plasmid-free and wild-type chlamydial organisms. CBA/J (n = 5) intravaginally inoculated with Pf C. muridarum (clone CMUT3.5) were intragastrically coinoculated with Wt Chlamydia (CMpmCherry) 7 days after intravaginal inoculation (see the Fig. 4 legend above). On day 28 after intravaginal inoculation, mice were sacrificed for recovering live organisms (black bar for Pf and red bar for Wt log₁₀ IFU along the y axis) from both genital (vagina/cervix [V], uterine horn [UH], and oviduct and ovary [O]) and GI (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) tissues (x axis).

FIG 8

Monitoring chlamydial genomes from mice coinoculated with both plasmid-free and wild-type chlamydial organisms. Tissue samples from the same CBA/J mice (n = 5) intravaginally inoculated with Pf C. muridarum and intragastrically coinoculated with wild-type (red) Chlamydia 7 days after intravaginal inoculation as described in the Fig. 7 legend were also used for detecting chlamydial DNA using qPCR. The results are expressed as the log₁₀ chlamydial 16S rRNA (black bars) or mCherry (red bars) gene copy numbers, as shown along the y axis. Genital tissues include vagina/cervix (V), uterine horn (UH), and oviduct and ovary (O), while GI tissues include stomach, duodenum, jejunum, ileum, cecum, colon, and rectum tissues, as listed along the x axis.