CONICET | Buscador de Institutos y Recursos Humanos

Chlamydia trachomatis (CT) is an obligate intracellular pathogen and the leading cause of sexually transmitted bacterial infections globally. CT resides inside a vacuole or ?inclusion? and undergoes a life cycle involving the infectious elementary body and the replicating reticulate body. Polymorphic membrane proteins (PMPs) are a family of Chlamydia-specific autotransporter proteins with proposed roles in adhesion and antigenic variation. However, due to technical limitations in genetic manipulation of this bacterium, the role of PMPs in CT biology remains poorly elucidated. By chemical mutagenesis coupled with whole genome sequence analysis, we obtained 3 mutants with nonsense mutations in pmpA/B/C. Due to the presence of extra mutations/genome (pmpA: 6; pmpB: 17;pmpC: 3) we backcrossed these mutants with a Wt strain to obtain recombinants carrying only the mutations in pmp genes. We obtained cleaned recombinants for pmpA/C. For pmpB mutant we were able to clean 14 of 17 mutations. We carried out IFU (Infectious Forming Units) assays in HeLa cells and observed that the generation of infectious progeny in the recombinants carrying nonsense mutations for pmpA/B/C was significantly impaired compared to Wt. These data point out that PMPs are important for efficient propagation of CT in epithelial cells. Additionally, by confocal microscopy and electron microscopy, pmpC strain showed an aggregation phenotype that could not be trans-complemented with the Wt strain in coinfection assays, suggesting a role for PmpC in preventing aggregation of individual bacteria inside the chlamydial inclusion. We are currently using a recently developed technology for gene deletion in CT by insertion of a group II intron in order to obtain pmpA, pmpB and pmpC null mutants to evaluate the role of PMPs in CT pathogenesis in a mice model of infection.