Wellslager, B.; Roberts, J.; Chowdhury, N.; Madan, L.; Orellana, E.; Yilmaz, O.

Porphyromonas gingivalis, a major oral pathobiont, evades canonical host pathogen clearance in human primary gingival epithelial cells (GECs) by initiating a non-canonical variant of autophagy consisting of Microtubule-associated protein 1A/1B-light chain 3 (LC3)-rich autophagosomes, which then act as replicative niches. Simultaneously, P. gingivalis inhibits apoptosis and oxidative-stress, including extracellular-ATP (eATP)-mediated reactive-oxygen-species (ROS) production via phosphorylating Heat Shock Protein 27 (HSp27) with the bacterial nucleoside-diphosphate-kinase. Here, we have mechanistically identified that P. gingivalis-mediated induction of HSp27 is crucial for the recruitment of the LC3 isoform, LC3C, to drive the formation of live P. gingivalis-containing Beclin1-ATG14-rich autophagosomes that are redox sensitive and non-degrading. HSp27 depletions of both infected GECs and gingiva-mimicking organotypic culture systems resulted in the collapse of P. gingivalis-mediated autophagosomes, and abolished P. gingivalis-induced LC3C-specific autophagic-flux in a HSp27-dependent manner. Concurrently, HSp27 depletion accompanied by eATP treatment abrogated protracted Beclin 1-ATG14 partnering and decreased live intracellular P. gingivalis levels. These events were only partially restored via treatments with the antioxidant N-acetyl cysteine (NAC), which rescued the cellular redox environment independent of HSp27. Moreover, our functional cellular-molecular findings, supported by our in-silico predictions, critically show that the temporally phosphorylated HSp27 tightly partners with LC3C, hindering LC3C canonical cleavage, extending Beclin 1-ATG14 association, and halting canonical maturation. These findings pinpoint how HSp27 pleiotropically serves as a major platform-molecule, redox regulator, and stepwise modulator of LC3C during P. gingivalis-mediated non-canonical autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adapted P. gingivalis successful mucosal colonization and oral dysbiosis.