31Immunometabolism and regulation of mitochondrial reactive oxygen species (mROS) control the 32 immune effector phenotype of differentiated macrophages. Mitochondrial function requires 33 dynamic fission and fusion, but whether effector function is coupled to altered dynamics during 34 bacterial responses is unknown. We show that macrophage mitochondria undergo fission after 12 35 h of progressive ingestion of live Streptococcus pneumoniae (pneumococci), without evidence of 36Drp-1 phosphorylation at S616. Fission is associated with progressive reduction in oxidative 37 phosphorylation but increased mROS generation. Fission is enhanced by mROS production, 38 PI3K signaling and by cathepsin B, but is independent of inflammasome activation or IL-1 39 generation. Inhibition of fission reduces bacterial killing. Fission is associated with Parkin 40 recruitment to mitochondria, but not mitophagy. Fission occurs upstream of apoptosis induction 41 and independently of caspase activation. During macrophage innate responses to bacteria 42 mitochondria shift from oxidative phosphorylation and ATP generation to mROS production for 43 microbicidal responses by undergoing fission. 44 45 46 47 48
Author summary 52Changes in metabolism regulate function in immune cells, including macrophages which are key 53 cells in pathogen clearance. Mitochondria are cellular organelles that generate energy during 54 metabolism but also mitochondrial reactive oxygen species (mROS) that contribute to bacterial 55 killing. Mitochondria are dynamic organelles that form complex networks with varying degrees 56 of fragmentation or fusion, but the functional consequences of these processes on macrophage 57 function during bacterial infection are unknown. We show that sustained ingestion of live 58 bacteria triggers mitochondrial fragmentation, reducing metabolism but enhancing mROS 59 generation. Mitochondrial fragmentation is not part of a clearance pathway for damaged 60 mitochondria and is initiated before signs of cell death. Macrophage signalling pathways 61 activated during infection, and mROS generation, enhance mitochondrial fragmentation, and 62 inhibition of pathways promoting fragmentation reduces bacterial killing. Overall, these findings 63 suggest that responses to ingested bacteria trigger mitochondrial fragmentation, allowing 64 mitochondria to switch from energy generation during metabolism to organelles facilitating 65 bacterial killing. 66 67 68 130
Mitochondrial fission is a response to live bacteria 131Prior reports have also described Drp-1 independent mitochondrial fission in HeLa cells 132 containing Listeria monocytogenes [23]. In this model fission was induced by the cholesterol-133 dependent cytolysin (CDC), listeriolysin O, as well as by related CDCs, including pneumolysin 134
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