8. INTEGRITY OF THE PREDICTED PROTEASE MOTIF OF CTL0175/PTR IS REQUIRED FOR Chlamydia trachomatis RECOVERY FROM IFNGAMMA-INDUCED PERSISTENCE.

BETTUCCI FERRERO, GLORIA N.; PANZETTA, MARÍA E.; VERAGUAS HERRAN, SOFÍA; ANNA, AILEN N.; 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 frequent sexually transmitted bacterial pathogen and a common cause of asymptomatic, persistent infections leading to serious complications and negative outcomes in young women’s reproductive health. CT displays an obligate intracellular lifestyle involving the infectious elementary body (EB) and the replicative and non-infectious reticulate body (RB). Infection begins when EBs attach to epithelial cells and get internalized within a membrane-bound intracellular vacuole or “inclusion”. At early times post-infection, EBs transition into metabolically active and dividing RBs. At mid-cycle, upon impending release of the bacteria to the extracellular environment, RBs asynchronously transition back to EBs. If during replication CT is exposed to antimicrobial stimuli such as those triggered by the host cytokine gamma-interferon (IFNγ), these bacteria undergo a transient interruption in its replication cycle and enter into a viable but non-cultivable, “persistent” state, which can last for long periods of time. Upon removal of the stressor, CT is able to resume normal replication and developmental transitions to complete the interrupted cycle. The ability to undergo persistence is considered critical for CT pathogenesis, however, little is known about the molecular basis of this phenomenon. Previous findings from our laboratory demonstrated that the uncharacterized locus ctl0175/ptr, is required for RB to EB transition and recovery from IFN-induced persistence. Bioinformatic analysis indicates that ctl0175/ptr encodes a 108 KDa protein showing 23% homology with Escherichia coli zinc-dependent protease Ptr, whose protease activity has been linked to the presence of the conserved motif HXXEH in the N-terminal region of the predicted protein. Interestingly, CT Ptr contains the protease conserved motif HX(F)X(T)EH. To assess if Ptr protease activity may be involved in CT recovery from IFN-induced persistence, we generated an E108Q point mutant version of Ptr (ptrE108Q) by site-directed PCR mutagenesis on a CT expression plasmid encoding full length wild type ptr (ptrFLAG). Next, we transformed these plasmids into a ctl0175/ptr null CT serovar LGV-L2 background (L2 ptr::GII), obtaining L2 ptr::GII-PtrFLAG and L2 ptr::GII-PtrE108Q complemented strains. Using a self-generated anti-Ptr antibody, we verified that L2 wild type and complemented strains were able to express Ptr. Then, we carried out IFN-induced persistence experiments using the mentioned strains and quantified the generation of infectious progeny upon removal of IFN (recovery assay) by means of inclusion-forming units quantification. Interestingly, we found that while ptrFLAG rescued L2 ptr::GII defective recovery, ptrE108Q did not. Overall, our findings suggest that protease activity of Ptr mediates rapid recovery of CT upon IFN-induced persistence and contribute to elucidate Ptr’s role in CT persistence and pathogenesis.