Polymorphic membrane protein C (PmpC) participates in Chlamydia trachomatis persistence and invasion and prevents chlamydial autoaggregation.

PANZETTA, MARÍA E.; ANNA, AILEN NAHIR; BETTUCCI FERRERO, GLORIA NAZARENA; BASTIDAS, ROBERT J.; VALDIVIA, RAPHAEL H.; SAKA, HECTOR A.

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

SAIB - SAMIGE

Chlamydia trachomatis (CT) is the most frequent causative agent of bacterial sexually transmitted infections worldwide. CT is an obligate intracellular pathogen presenting a biphasic life cycle that involves the transition between infectious elementary bodies (EBs) and replicative but not infectious reticulate bodies (RBs). The cyclic transitions between EBs and RBs occur inside a CT-induced intracellular vacuole or ?inclusion?. In the presence of stressors such as beta-lactams or interferon-gamma (IFNg), CT enters into a poorly studied viable but non-cultivable state termed ?chlamydial persistence?, which is reversible upon removal of the stressors and considered critical for pathogenesis. Polymorphic membrane proteins (PMPs) are a family of Chlamydia-specific autotransporter proteins secreted via a type V secretion system. The genome of CT encodes 9 PMPs (PMPA-I), which have been proposed to play a role in antigenic variation and adherence, however, PMPs functions remain ill-defined due to Chlamydia being historically refractory to traditional genetic manipulation. In a previous screen with a collection of ~1000 genome sequenced CT chemical mutants, we identified a PMPC nonsense mutant (pmpC-ns) with a defective phenotype in chlamydial persistence. In order to confirm the role of PMPC in chlamydial persistence, a PMPC-null mutant was obtained via insertional gene inactivation with a group II intron (pmpC::GII). We observed that in control conditions, both wild type (WT) and pmpC::GII CT were able to complete their life cycle and generate similar amounts of infectious EBs. However, upon penicillin- or IFNg-induced persistence, pmpC::GII presented a defective phenotype, consistently showing a decreased production of EBs after removal of the persistence inducers. To further investigate PMPC functions in CT, adherence and invasion assays were carried out in epithelial HeLa cells using fluorescently-labeled WT, pmpC::GII and pmpC-ns CT. We found no statistically significant differences in adherence to HeLa cells between either strain. Nevertheless, pmpC::GII and pmpC-ns CT invasion rates were more than 10 fold lower than that observed for WT CT. Curiously, both pmpC-ns and pmpC::GII displayed an altered phenotype inside the inclusion, characterized by a non-homogeneous distribution of the bacteria, which were instead observed forming ?aggregates?. By performing live-cell microscopy of HeLa cells infected with fluorescently labeled WT, pmpC::GII or pmpC-ns CT, we confirmed that lack of PMPC was associated with ?auto-aggregation? inside the inclusion, which was not rescued by co-infecting with the WT strain, thus suggesting that homotypic PMPC interactions might prevent this aggregation phenomenon. In conclusion, these results support that PMPC participates in penicillin- and IFNg-induced persistence and CT invasion but not adherence, and also in preventing auto-aggregation of the bacteria inside the inclusion.