PINK1/Parkin mitophagy and the mitochondrial unfolded protein response (UPRmt) are activated in response to mitochondrial dysfunction to maintain mitochondrial health. Neisseria gonorrhoeae-derived outer membrane vesicles (OMVs) can target mitochondria and cause mitochondrial dysfunction and apoptosis. The role of mitochondrial quality control pathways such as PINK1/Parkin mitophagy and the UPRmt in modulating mitochondria to regulate cellular responses to pathogenic bacterial OMV exposure is unclear. Here, we assessed whether OMVs activate mitochondrial quality control pathways using host cells deficient in Parkin and/or CHOP, which is a transcription factor involved in stress response pathways including the UPRmt.
To assess the role of mitochondrial quality control in OMV exposure, OMVs were isolated from Neisseria gonorrhoeae (MSAII) grown to mid-log phase (OD600 < 1) in GC broth then harvested via ultracentrifugation. Protein and particle concentrations were assessed using BCA and NTA (ZetaView), respectively. Primary bone marrow-derived macrophages (BMDMs) from wild-type, Parkin-/-, CHOP-/-, and Parkin-/-/CHOP-/- mice were differentiated and exposed to N. gonorrhoeae OMVs at 20 μg/ml. Live cell imaging, western blots probing for mitochondrial stress markers, and cytokine profiling were conducted. Preliminary data show a significant reduction in cell death at 72 hours after OMV treatment in the absence of Parkin and/or CHOP. Moreover, Parkin-/-, CHOP-/-, and Parkin-/-/CHOP-/- BMDMs exhibited altered cytokine profiles, with reduced expression of proinflammatory cytokines. This data highlights key differences between wild-type and knockout genotypes in response to N. gonorrhoeae OMV exposure, suggesting a potential role of mitochondrial quality control systems in regulating macrophage survival and innate immune responses.