Role of ROS detoxifying enzymes for bacterial survival
Killing of phagocytosed bacteria might occur via different mechanisms. Several S. aureus factors were shown to contribute to protection against intoxication by copper ((copXL, copA ) (Purves et al., 2018)), ROS (staphyloxanthin biosynthesis (Liu et al., 2008; Olivier et al., 2009)) or H202 (katA , (Cosgrove et al., 2007)). Inactivation of copA , copL , orkatA in the „non-toxic“ USA300 strain did not impact bacterial survival in THP-1 cells (Fig. 3A). Staphyloxanthin biosynthesis is strictly dependent on the alternative sigma factor B (SigB) (Mader et al., 2016) rendering sigB mutants non-pigmented. Since deletion of sigB did not alter bacterial survival in THP-1 cells, protection by staphyloxanthin or other SigB regulated factors is not essential for bacterial survival. In murine macrophages the GraRS regulon was shown to contribute to bacterial survival in USA300 wild-type bacteria (Flannagan et al. , 2018). However, in theagr/sae negative background no significant difference in intracellular survival was observed (Fig. 3A)
Interestingly, S. aureus possesses two sod genes,sodA and sodM , which is unique among Gram-positive bacteria (Valderas et al., 2002). Other staphylococci only possess one Sod homodimer resembling S. aureus SodA. Thus, we speculated that sodM may contribute to the unique survival of S. aureus in macrophages. But when we deletedsodA , sodM or both genes in the „non-toxic“ USA300 strain background, we did not observe a decrease in bacterial survival in THP-1 cells (Fig. 3B). However, THP-1 cells may produce less ROS compared to primary macrophages. We therefore tested the mutant strains for survival in hMDMs. In these cells the double mutant exhibited a significant decrease in survival rate after 24 h (Figure 3D). The LDH-assay confirmed that this decrease in CFU could not be attributed to better escape of the sodA-sodM -mutant (Figure 3E). We next overexpressedsodM in S. epidermidis using an anhydro-tetracycline inducible promoter. SodM expression could not rescue S. epidermidis . Thus, the mutant analyses could not link the better survival of S. aureus versus CoNS in THP-1 cells to any property to deal with ROS or toxic copper. In hMDMs S. aureus specific expression of two superoxide dismutases contributes to bacterial survival but cannot protect S. epidermidis from being killed.
Intracellular acidification is linked to killing of CoNS but not of „non-toxic“S. aureus
S. aureus resides and multiplies in mature phagolysosomes in murine and human macrophages (Flannagan et al. , 2015; Pidwillet al. , 2021). Low pH even promotes survival of USA300 whereas other S. aureus strains such as strain Newman seem to be sensitive to low pH (Flannagan et al. , 2018)(Tranchemontagne et al., 2015)(Sedlyarov et al. , 2018). We assayed whether „non-toxic“ S. aureus or CoNS strains were sensitive to the v-ATPase inhibitor bafilomycin, which inhibits lysosomal acidification (Sedlyarov et al. , 2018). The drug had no impact on bacterial growth when added to bacterial cultures (Fig. S1). Bacterial survival or cytotoxicity was not significantly altered in bafilomycin treated THP-1 cells indicating both „non-toxic“ S. aureus strains USA300 and Newman are insensitive towards pH alterations in the phagosomes. Survival of CoNS S. epidermidis was significantly increased in bafilomycin treated cells. However, S. epidermidis was not found to be more sensitive to pH when grown in medium (Fig. S2).