Complex-I Preserves Mitochondrial Polarization during Infection of Human Macrophages by Secretion-competent Bacteria

Intracellular bacteria remodel host bioenergetics and modulate mitochondrial membrane potential (Δψm). However, how individual electron-transport chain (ETC) components sustain Δψm during infection of primary human macrophages remains unclear. Here we combine extracellular flux analysis with single-cell live imaging to understand how the ETC functions in human monocyte-derived macrophages (hMDMs) during infection with ( Legionella pneumophila ( Lp ) or Salmonella enterica serovar Typhimurium ( S .Tm). At 5 h post-infection, the Lp type IV secretion system (T4SS) and the S .Tm SPI-1 T3SS were required for the early drop of the oxygen consumption rate. Despite reduced respiration, the Δψm was preserved in all infection conditions and pathogen-specific strategies to maintain the Δψm were revealed. While Lp infection modulates the F _O F ₁ -ATPase to function in the reverse mode (hydrolase) with the adenine-nucleotide translocator (ANT) remaining in forward mode, S .Tm does not reverse the F _O F ₁ -ATPase during infection. Systematic inhibition of ETC complexes established that Complex I is uniquely required to maintain the Δψm during infection with virulent bacteria but not with secretion-deficient mutant strains. Complex II is required in all infection conditions but its inhibition had a minimal effect in non-infected cells, indicating infection-driven participation of this complex in the electron flow in the ETC coupled with the preservation of the Δψm. Complexes III and IV were essential in infected and non-infected cells. Together, our results identify a Complex I-driven maintenance of the Δψm, establishing Complex I as a bioenergetic checkpoint that distinguishes virulent from secretion-deficient intracellular bacteria. Furthermore we reveal that divergent strategies are employed by Lp and S .Tm to preserve macrophage mitochondrial polarization early during infection.