Porphyromonas gingivalisactivates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

Abstract

ABSTRACTPorphyromonas 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. gingivalisinhibits 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 (Ndk). Here, we have mechanistically identified thatP. gingivalis-mediated induction of HSp27 is crucial for the recruitment of the LC3 isoform, LC3C, to drive the formation of liveP. 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 ofP. gingivalis-mediated autophagosomes, and abolishedP. 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 intracellularP. gingivalislevels. 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, the temporal phosphorylation of HSp27 by the bacterial Ndk results in HSp27 tightly partnering 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 duringP. gingivalis-mediated non-canonical autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adaptedP. gingivalis’ successful mucosal colonization and oral dysbiosis.