FIG 3.

Genetic rescue of CtD pgp3^– infectivity in female CRAMP KO mice and a molecular mechanism of Pgp3-mediated inhibition of AMP activity. (A) Genetic rescue of CtD pgp3^– infectivity in CRAMP gene-deficient mice. Ten female C57BL/6 or CRAMP KO mice were infected transcervically with 1 × 10⁵ IFU of CtD pgp3^– organisms, and infectious loads (rIFU) present in cervicovaginal swabs were determined at days 3 to 28 p.i. The P values were calculated with a Mann-Whitney test. (B) Immunofluorescence of HeLa229 cells infected with CtD, CtD P⁻, CtD pBRDUW3, and CtD pgp3^– at 40 h p.i. stained with anti-Pgp3 (red), anti-MOMP (green), and DAPI (blue). Arrows denote inclusion body and secreted cytosolic Pgp3 in CtD- and CtD pBRDUW3-infected cells. (C and D) Secreted extracellular Pgp3 inhibits AMP killing of chlamydiae. (C) CtD, CtD P⁻, CtD pBRDUW3, and CtD pgp3^– organisms were incubated with different concentrations of CRAMP for 1.5 h at room temperature, and infectivity was assayed by titration of IFU on McCoy cells. CRAMP inhibited infectivity of all strains in a dose-dependent manner but, importantly, was not Pgp3 dependent. The P values were calculated with Student’s t tests. (D) Supernatants from osmotically lysed cells infected with CtD, CtD P⁻, CtD pBRDUW3, and CtD pgp3^– at 40 h pi were incubated with different concentrations of CRAMP for 1.5 h at room temperature, and infectivity was assayed by titration of rIFU on McCoy cells. The infectivity of CtD and CtD pBRDUW3 was not significantly reduced at any concentration. In marked contrast, the infectivity of CtD P⁻ and CtD pgp3^– was significantly reduced in a dose-dependent manner following CRAMP treatment. Statistical significance by Student’s t test is indicated as follows: NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.