B28-03 Outer Membrane Vesicles Secreted by Pseudomonas Aeruginosa Promote Mitochondrial Transfer Through Activating Mitochondrial Connexin 43 in Airway Cells

Abstract Rationale Pseudomonas aeruginosa is a major health challenge that causes recalcitrant multi-drug-resistant infections, especially in immunocompromised and hospitalized patients. Multi-drug-resistant strains of P. aeruginosa are increasing threats contributing to high mortality in these patients and can lead to multi-organ failure. Bacterial outer membrane vesicles (OMVs) modulate local and systemic pro-inflammatory immune responses and facilitate intercellular communication by delivering virulence factors. Cell-to-cell transfer of mitochondria plays an important role in the progression of diseases. Gap junctions connexin 43 (Cx43) regulate mitochondrial function and mediate mitochondrial transfer. The molecular mechanisms by which OMVs modulate innate immunity and mitochondrial transfer during infections are not well defined. We and others have previously shown that P. aeruginosa leads to mitochondrial dysfunction in epithelial cells, affecting tight junction and cellular metabolism. In this study, we elucidated the protein profile of OMVs and determined the effect of Cx43-based mitochondrial function in P. aeruginosa-infected epithelial cells. Methods OMVs from P. aeruginosa PABLO clinical strain bacteria were isolated and characterized by Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM). The global proteome profile of OMVs was analyzed by LC-MS/MS and Spectronaut software. Target gene expression levels were analyzed using quantitative RT-PCR, Western blot, and ELISA. The gain or loss of target genes was performed using CRISPR/Cas9 gene editing. Mitochondrial bioenergetics were verified through a mitostress test and ATP production assay, as well as a glycolysis assay. Results Over four hundred proteins in P. aeruginosa-OMVs were identified with high confidence by LC-MS/MS analyses. Proteomic analysis revealed diverse lipid species and proteins potentially involved in regulating metabolic dysfunction. Inflammatory cytokines were increased by OMVs from P. aeruginosa, which enhanced junctional gap communication, with increased expression levels of Cx43 and Cx45 in airway epithelial cells. A metabolic bioenergetics analysis test revealed that OMVs impaired mitochondrial oxidative phosphorylation in airway epithelial cells. Targeting the Cx43 modulated genes regulating mitochondrial biogenesis in airway epithelial cells. CRISPR interference reduced expression of Cx43 and restored mitochondrial oxidative phosphorylation and ATP release in epithelial cells exposed to OMVs. Cx43 silencing suppressed inflammatory cytokine secretion and restored the SIRT1-PGC-1α-TFAM signaling pathway. Conclusions OMVs from P. aeruginosa PABLO strain activate the inflammatory pathways and a regulator of mitochondrial transfer Cx43 and modulate mitochondrial biogenesis via the SIRT1/PGC-1alpha/TFAM in airway epithelial cells. These findings provide novel insights into the function of the mitochondrial Cx43 in airway epithelial cells during P. aeruginosa infections and a therapeutic approach for resistant infections. This abstract is funded by: I01BX001786; R01HL144478