„Non-toxic“ S. aureus survives better in macrophages than CoNS
The virulence regulators Agr and Sae control the synthesis of most immunomodulatory molecules or toxins such as LukAB or phenol-soluble modulins (PSMs) and a „non-toxic“ agr/sae mutant is unable to escape after phagocytosis (Münzenmayer et.al, 2016). We compared survival of wild type and „non-toxic“ S. aureus within THP-1 cells with that of various CoNS isolates (multiplicity of infection (MOI =10)). More than 90% of the inoculated bacteria were phagocytosed (Fig. 1A). Within 24 h, the cytotoxic USA300 escaped the macrophages. This is indicated by a severe decrease in colony forming units (CFUs) after 24 h (Fig. 1A) as the escaped bacteria were efficiently killed by gentamycin. However, the „non-toxic“ USA300 was retained in the THP-1 cells at high numbers. CoNS showed a significant decrease in CFU compared to the „non-toxic“ USA300. Especially S. pettenkoferi was not able to survive intracellularly. To analyse whether the decrease in CFU after 24 h was due to escaped or intracellularly killed bacteria we performed a cytotoxicity assay. After 24 h lactate dehydrogenase (LDH) release, a proxy for host cell lysis, was only observed after uptake of USA300 wild-type bacteria. No cytotoxicity was detectable after phagocytosis of „non-toxic“ USA300 or CoNS strains (Fig. 1B) indicating that the CoNS species are indeed killed after phagocytosis.
We followed the intracellular bacteria by live-cell imaging using the THP-1-CWT cell line expressing the S. aureus recruitment marker YFP-CWT, which recognizes peptidoglycan (Grosz et al., 2014). YFP-CWT cannot enter the phagosome and thus recognizes only cytosolic bacteria by binding to the bacterial surface. The escape from phagosome and cell-death of the host macrophage were observed in USA300 infected cells (Supplemental movie S2). However, internalization of the „non-toxic“ USA300 agr/sae mutant or the S. epidermidis strain did not result in obvious cell damage (Supplemental movie S3 and S4). We quantified live intracellular bacteria using Syto9 staining. Even after 48 h, the number of macrophages harbouring live S. aureus did not decrease. However, the number of infected macrophages harbouring liveS. epidermidis dropped significantly indicating that S. epidermidis was cleared in a part of the macrophage population (Figure 2E). The bacterial number per staphylococcal positive macrophages was also significantly lower in CoNS infected macrophages and was further decreased after 48 h. Thus, „non-toxic“ S. aureus can survive phagocytosis whereas a large part of the CoNS bacteria is cleared from macrophages.