Neutrophil Extracellular Mitochondria Are Endowed With Direct Bacteriostatic/Bactericidal Activity on Staphylococcus...

Neutrophils are the most abundant type of leukocyte found in human peripheral blood, with approximately 1 × 10¹¹ produced daily in the bone marrow. They play a crucial role as the first line of defence against microbial infections. Neutrophils utilise various mechanisms to combat pathogens, including phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). Upon exposure to bacteria, neutrophils may release some of their mitochondria into the extracellular environment, which then tag bacteria. This process aids in the phagocytosis of the bacteria, enhancing their elimination within the neutrophil's phagolysosomes. According to the endosymbiotic theory, mitochondria originated from an alpha-proteobacterium. Since many bacteria possess antimicrobial mechanisms that enable them to survive in resource-limited ecosystems, we inquired whether mitochondria might have retained or developed direct bacteriostatic/bactericidal capabilities. Mitochondria were extracted from neutrophils, and Staphylococcus aureus was selected as the bacterial target. Bacterial growth was assessed using Colony Forming Units (CFU) counts and turbidimetry (optical density) in cultures containing only bacteria or bacteria combined with extracellular mitochondria; bacterial LIVE/DEAD assays were also performed. The interaction between mitochondria and bacteria was analysed using scanning and transmission electron microscopy. The results demonstrated that the presence of mitochondria reduced bacterial growth in culture; however, LIVE/DEAD assays showed that most bacteria remain viable. Together, these findings suggest that extracellular mitochondria mostly induce a viable but non-culturable (VBNC) state in S. aureus. The interaction between mitochondria and bacteria was strong, leading to morphological changes in the bacteria and, in some instances, this interaction ultimately caused the disruption of the bacterial cell wall and the loss of intracellular contents. Morphological changes were also observed in the mitochondria, including the formation of vesicular structures. Exposure of the mitochondria to MitoQ, a potent mitochondrial reactive oxygen species (mROS) inhibitor, partially reversed its effect on S. aureus CFU counts but had no effect on viability or the interaction between mitochondria and bacteria. Our findings suggest that cell-free extracellular mitochondria possess the potential for direct bacteriostatic and, to a lesser extent, bactericidal activity, which is at least partially mediated by mROS.