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).