Chlamydia trachomatis POLYMORPHIC MEMBRANE PROTEIN C PARTICIPATES IN DEVELOPMENTAL TRANSITIONS DURING BACTERIAL RECOVERY FROM INTERFERON GAMMA AND PENICILLIN-INDUCED PERSISTENCE

ANNA, AILEN N.; PANZETTA, MARÍA E.; BETTICCI FERRERO, GLORIA NAZARENA; VERAGUAS HERRAN, SOFÍA; SAKA, H. ALEX

Rosario

Congreso; LIX Congreso anual de la Sociedad Argentina de Bioquímica y Biología Molecular ? SAIB; 2023

Sociedad Argentina de Bioquímica y Biología Molecular ? SAIB

Chlamydia trachomatis (CT) is the most prevalent etiologic agent of bacterial sexually transmitted infections worldwide. CT infection is a frequent cause of asymptomatic and persistent infections specially in young women, leading to severe complications like pelvic inflammatory disease, infertility, and ectopic pregnancy. CT is an obligate intracellular pathogen with a unique lifestyle involving two developmental forms, the infectious elementary body (EB) and the replicative but non-infectious reticulate body (RB). EBs first adhere to epithelial cells and then get confined within an intracellular vacuole or “inclusion”, to rapidly transition into replicative RBs. Around mid-cycle, RBs transition back to EBs and bacteria are released to the extracellular space to reinitiate the infectious cycle. When exposed to stressors such as gamma-interferon (INF) or penicillin antibiotics, CT halts replication and enters a viable but non-cultivable state called chlamydial persistence. Upon removal of stressors, CT resumes replication. Previous results from our group identified that members of a Chlamydia-specific family of autotransporter proteins called Polymorphic Membrane Proteins (Pmps), are implicated in persistence. A pmpC-null mutant (L2 pmpC::GII) confirmed that PmpC is required for CT recovery from INF- and penicillin-induced persistence. To assess the mechanistic basis of impaired recovery, we carried out electron microscopy analysis of CT-infected cells and evaluated the content of developmental forms. No differences were found between L2 pmpC::GII compared to L2 wild type strain in the untreated condition. However, during recovery from both, IFNγ- and penicillin-induced persistence, L2 pmpC::GII inclusions show fewer EBs and a reduced EB/RB ratio. This indicates that lack of PmpC negatively affects RB to EB transition, which in turn explains impaired generation of infectious progeny upon recovery. To further investigate the role of PmpC in CT persistence and pathogenesis, two different PmpC fragments were expressed in Escherichia coli, tag-purified, and used to generate PmpC polyclonal antibodies, which were then successfully used in immunoblots and immunofluorescence assays. PmpC was found to be expressed throughout mid to late stages of the CT life cycle. Interestingly, PmpC-specific fluorescent signal was found partially not co-localizing with the bacteria body both in untreated and recovery conditions. Moreover, fractionation assays of CT-infected HeLa cells carried out to separate bacterial (pellet) and host cells (supernatant) showed that PmpC was detected in both fractions. Finally, by using the two previously mentioned anti-PmpC antibodies, we found evidence of post-translational cleavage between Ser-564 and Gly-596. Overall, these findings indicate that PmpC is proteolytically processed, secreted to the lumen of inclusions and required for efficient RB to EB transition during recovery from INF- and penicillin-induced persistence.