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 combined extracellular flux analysis with single-cell live imaging to understand how the ETC functions in human monocyte-derived macrophages during infection with <i>Legionella pneumophila</i> (<i>Lp</i>) or <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>.Tm). At 5 h post-infection, the <i>Lp</i> type IV secretion system (T4SS) and the <i>S</i>.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 <i>Lp</i> infection modulates the F_OF₁-ATPase to function in the reverse mode (hydrolase), with the adenine-nucleotide translocator remaining in forward mode, <i>S</i>.Tm does not reverse the F_OF₁-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 <i>Lp</i> and <i>S</i>.Tm to preserve mitochondrial polarization of macrophages early during infection.