Characterization of Chlamydia trachomatis Polymorphic Membrane Protein C Biological Functions During Persistence and Infection of Epithelial Cells

ANNA, AILEN NAHIR; PANZETTA, MARÍA E.; BETTUCCI FERRERO, GLORIA NAZARENA; MARIN, C.; MALETTO, BELKYS; VALDIVIA, RAPHAEL H.; SAKA, HÉCTOR A.

Buenos Aires

Taller; VIII Taller Internacional de Infecciones por Chlamydiales en Humanos y Animales; 2022

Facultad de Farmacia y Bioquímica, UBA

Introduction: Chlamydia trachomatis (CT) is capable of evading antimicrobial stimuli like those triggered upon exposure to beta-lactams and interferon-gamma (IFNg), by entering into a reversible non-cultivable state termed “chlamydial persistence” or “Chlamydia stress response”. Although this process is considered relevant for pathogenesis, the chlamydial genes involved are poorly elucidated. Material and methods: we carried out a high-throughput screening in HeLa cells using a fully sequenced collection of ~1000 chemical CT mutants in order to identify chlamydial genes required for recovery upon IFNg- and penicillin-induced persistence. Results: 5 out of 8 CT mutants exhibiting a defective persistence phenotype for either stressor, harbored mutations in Polymorphic Membrane Proteins (Pmps), a Chlamydia-specific family of immunogenic autotransporter proteins likely involved in adherence to epithelial cells. We focused our analysis on mutant M111, presenting a nonsense mutation on pmpC along with 8 additional mutations. A pmpC-null mutant (pmpC::GII) obtained by targeted group II-intron mutagenesis in a CT L2 434/Bu genetic background devoid of additional mutations, further confirmed that this gene was required for efficient recovery upon both, IFNg- and penicillin-induced persistence. Two different PmpC fragments (rPmpC1 and rPmpC2) were recombinantly expressed in Escherichia coli, His-tag-purified and used to generate PmpC polyclonal antibodies in mice. Both antibodies were successfully used in immunoblots and immunofluorescence assays, confirming that pmpC::GII does not express PmpC. We took advantage of the pmpC::GII mutant to further investigate the biological functions of PmpC during infection, and carried out adherence and invasion assays. We found that while PmpC is dispensable for CT adherence, lack of PmpC significantly impaired invasion to HeLa cells. Additionally, by confocal fluorescence microscopy we analyzed the expression pattern of PmpC at mid and late stages of the CT life cycle in untreated, as well in IFNg- or penicillin-induced persistence and recovery. We found that PmpC is expressed all along the CT infection cycle regardless of the presence/absence or post-removal of IFNg and pencillin. Intersingly, PmpC-specific fluorescent signal not co-localizing with the bacteria suggested that PmpC is secreted to the lumen of the chlamydial inclusion, being this observation particularly evident at mid-cycle. Of note, we observed that pmpC::GII displayed a non-homogeneous distribution of the bacteria within the inclusion, resembling bacterial “aggregates”. Next, we carried out live-cell microscopy of HeLa cells infected with fluorescently labeled WT, pmpC::GII or M111 CT strains, and found that the “aggregation” phenotype was not rescued by co-infecting with the WT strain, pointing out that PmpC homotypic interactions might be required to prevent aggregation. Lastly, by using the two previously mentioned mice-derived anti-PmpC antibodies in immunoblot assays, which recognize different portions of this protein, we clearly observed evidence of post-translational cleavage that may take place between Ser-564 and Gly-596. Discussion: the results presented herein uncover a novel role for PmpC in Chlamydia persistence and explore the possible consequences of PmpC abrogation in virulence-relevant CT properties such as adherence and invasion to epithelial cells. While not significantly affecting adherence, lack of PmpC results in a marked decrease of bacterial invasion. Also, we present evidence that PmpC is proteolytically processed, secreted to the inclusion lumen and that this protein is required to prevent auto-aggregation likely via homotypic interactions.