The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract

Abstract

Despite the extensive in vitro characterization of CPAF (chlamydial protease/proteasome-like activity factor), its role in chlamydial infection and pathogenesis remains unclear. We now report that a Chlamydia trachomatis strain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficient C. trachomatis strain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promoting C. trachomatis survival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.

FIG 1

Effect of CPAF deficiency on chlamydial survival/infectivity in the lower genital tracts of mice following an intravaginal inoculation. Seven-week-old C3H/HeJ female mice were each inoculated with 5 × 10⁶ inclusion-forming units (IFUs) of L2-17 (panel a, n = 15), L2-5 (panel b, n = 8), or L2wt (panel c, n = 15) intravaginally. On different days after inoculation as indicated along the x axis, vaginal swabs were taken for monitoring recovery of live chlamydial organism. The recovered live organisms were expressed as log₁₀ IFUs per swab and presented individually (open circles) along the y axis. Each horizontal bar represents the median for each group. The data came from 3 independent experiments. Note that mice intravaginally inoculated with L2-17 displayed a significantly lower level of live-organism recovery than the mice similarly infected with L2-5 or L2wt (Wilcoxon; **, P < 0.01).

FIG 2

Effect of CPAF deficiency on inflammatory infiltration in the genital tracts of mice following an intravaginal inoculation. (A) The mice described in the Fig. 1 legend were sacrificed on days 21 to 60 postinfection, and the genital tracts were processed for microscopic detection of inflammatory infiltration. Five mice from each group sacrificed on or close to day 21 were selected for the pathology analysis. The mouse sacrifice dates were matched among the 3 groups. Representative images of uterine horn tissue from each group of mice were taken under 10× (a to c) and 100× (a1 to c1) objective lenses. The areas covered under the 100× lens are marked with white rectangles in the corresponding 10× images. (B) Different sections of the genital tract, including the lumen (Lum) and tissue (Tis) of vagina (Va), uterine/uterine horns (Ut), and oviducts (Ov), were semiquantitatively scored for inflammatory infiltration based on the criteria described in Materials and Methods. The inflammatory scores from each mouse were plotted individually along the y axis. Solid circles stand for the inflammatory scores from Va-Lum, solid squares for scores from Va-Tis, solid standard triangles for scores from Ut-Lum, solid inverted triangles for scores from Ut-Tis, solid diamonds for scores from Ov-Lum, and open circles for scores from Ov-Tis. Note that the inflammatory scores indicated for the uterine/uterine horn tissues (Ut-Tis) of mice intravaginally inoculated with L2-17 (a) are significantly lower (Wilcoxon; *, P < 0.05) than those indicated for mice similarly inoculated with either L2-5 (b) or L2wt (c).

FIG 3

Effect of CPAF deficiency on chlamydial survival in the lower genital tracts of mice following an intrauterine inoculation. Seven-week-old C3H/HeJ female mice were each infected with 5 × 10⁶ IFUs of L2-17 (panel a; n = 10), L2-5 (panel b; n = 5), or L2wt (panel c; n = 10) via an intrauterine inoculation. On different days after inoculation as indicated along the x axis, vaginal swabs were taken for monitoring recovery of live chlamydial organisms. The recovered levels of live organisms are expressed as the number of log₁₀ inclusion-forming units (IFUs) per swab as shown along the y axis. No significant difference in levels of live-organism shedding was found between the groups (Wilcoxon).

FIG 4

Effect of CPAF deficiency on inflammatory infiltration in the mouse genital tracts following an intrauterine inoculation. (A) The mice described in the Fig. 3 legend were sacrificed on days 21 to 60 postinfection, and the genital tracts were processed for microscopic detection of inflammatory infiltration. Representative images of uterine horn tissue from each group of mice were taken under 10× (a to c) and 100× (a1 to c1) objective lenses. The areas covered under the 100× lens are marked with white rectangles in the corresponding 10× images. (B) Different sections of the genital tract, including the lumen (Lum) and tissue (Tis) of vagina (Va), uterine/uterine horns (Ut), and oviducts (Ov), were semiquantitatively scored for inflammatory infiltration based on the criteria described in Materials and Methods. The inflammatory scores from each mouse were plotted individually along the y axis. Solid circles stand for the inflammatory scores from Va-Lum, solid squares for scores from Va-Tis, solid upright triangles for scores from Ut-Lum, solid upside triangles for scores from Ut-Tis, solid diamonds for scores from Ov-Lum, and open circles for scores from Ov-Tis. No significant differences in inflammatory infiltration scores were found between the groups (Wilcoxon).

FIG 5

CPAF-dependent chlamydial survival in the lower genital tracts of multiple strains of mice. Six-week-old female C3H/HeJ (a and b; n = 5 for each group), C57BL/6J (c and d; n = 5), and BALB/cJ (e and f; n = 5) mice were intravaginally inoculated with 10⁶ IFUs of L2-17 (a, c, and e) or L2-5 (b, d, and f). Vaginal/cervical swabs were taken on different days after the inoculation as indicated along the x axis for monitoring live-organism recovery as shown along the y axis. Note that the L2-17 organisms displayed significantly lower levels of live-organism recovery than the L2-5 organisms regardless of the strains of mice infected (Wilcoxon; **, P < 0.01).

FIG 6

CPAF complementation effect on chlamydial survival in mouse lower genital tract. Seven-week-old C57BL/6J female mice were each inoculated with 1 × 10⁶ IFUs of L2-17 (a; n = 5), L2-17/CPAF (b; n = 5), or L2-17/mCherry (c; n = 5) intravaginally. On different days after inoculation as indicated along the x axis, vaginal swabs were taken for monitoring recovery of live chlamydial organisms. The recovered live organisms were expressed as log₁₀ inclusion-forming units (IFUs) per swab as shown along the y axis. Note that L2-17/CPAF developed more-extensive live-organism shedding than either L2-17 or L2-17/mCherry (P < 0.01 or P < 0.05, respectively; Wilcoxon).