Plasmid-encoded Pgp3 protein enhances the infectivity of Chlamydia trachomatis serovar L2 in mice.
Sun Xin X, Zhang Qi Q, Tian Qi Q, Xue Min M et al.
Chlamydial infection can cause a variety of serious diseases, including trachoma, tubal infertility, and rectal lymphogranuloma venereum (LGV). However, the underlying pathogenic mechanisms remain incompletely understood. Chlamydia trachomatis (C. trachomatis) serovar L2 was among the first chlamydial strains to be successfully transformed and has been widely used to investigate the functions of chlamydial proteins, particularly plasmid-encoded proteins. Previous studies of serovar L2 have largely been limited to in vitro experiments, and the roles of plasmid-encoded proteins during in vivo infection remain poorly understood. This study aims to evaluate the infectivity in mice of L2 mutants carrying deletions or premature stop codon mutations in different plasmid genes. Female C3H/HeJ and CBA/J mice received a subcutaneous injection of medroxyprogesterone acetate 5 days before infection and were subsequently inoculated intravaginally or intrauterinely with 1.0×106 inclusion-forming units (IFUs) of various L2 strains. These included wild-type L2 (L2wt), plasmid-free L2 (L2R), green fluorescent protein (GFP)-expressing L2 (L2GFP), plasmid-encoded glycoprotein (Pgp)-related deletion mutants (L2ΔPgp3, L2ΔPgp4, L2ΔPgp5, and L2ΔPgp7), and premature stop-codon mutants (L2Pgp3Y9S, L2Pgp4K13S, L2Pgp5L9S, L2Pgp7L11S, and L2Pgp8E11S). Vaginal swabs were collected at regular intervals after infection, and viable chlamydial shedding was monitored by immunofluorescence-based titration. The infectivity of each mutant was compared with that of the L2wt or L2GFP control group. For in vitro experiments, HeLa cells infected with the above strains were harvested at 30 or 48 h post-infection. Immunofluorescence staining and Western blotting (WB) were performed to assess the expression levels of Pgp3 and glycogen synthase A (GlgA), respectively. Compared with L2wt, the plasmid-free strain showed significantly reduced chlamydial shedding after intrauterine infection (P<0.05), whereas no such difference was observed after intravaginal infection, suggesting that the L2 plasmid plays a more important role in upper genital tract infection than in lower genital tract infection. Deletion of either Pgp3 (L2ΔPgp3) or Pgp4 (L2ΔPgp4) resulted in significantly reduced vaginal shedding of viable chlamydia after intrauterine inoculation (P<0.05). Mice infected with the Pgp3 premature stop codon mutant L2Pgp3Y9S exhibited reduced shedding (P<0.05), whereas this phenotype was not observed with the Pgp4 mutant L2Pgp4K13S. Immunofluorescence staining and WB showed that L2Pgp4K13S still expressed a detectable amount of Pgp3, although markedly less than L2wt, and this residual expression may have been sufficient to maintain infectivity. WB further confirmed that expression of the C. trachomatis glycogen synthase GlgA depends on Pgp4 rather than Pgp3. The infectivity of the Pgp4-deficient strain L2Pgp4K13S was comparable to that of L2wt, suggesting that GlgA may not be required for L2 infection of the murine upper genital tract. Pgp3 rather than Pgp4, other plasmid-encoded genes, or the plasmid-regulated GlgA gene, plays a critical role in the infectivity of C. trachomatis serovar L2 in mice. In addition, even very low levels of Pgp3 expression may be sufficient to maintain L2 infectivity in mice.