Discussion
Macrophages are important professional phagocytes to combat infections. However, from in vitro and in vivo studies, it is evident that they fail to eradicate S. aureus (Hommes and Surewaard, 2022; Pidwillet al. , 2021). S. aureus is provided with an impressive arsenal of virulence determinants that give pathogenic potential to this bacterium. The intracellular expression of virulence factors is to a large extent coordinated via the two-component regulatory systems Agr (Bronesky et al., 2016) and Sae (Liu et al. , 2016). Induction of pore-forming toxins such as the Sae regulated LukAB and the Agr regulated PSMs are important for the escape of the bacteria from the intracellular environment within macrophages (Melehani et al., 2015; Munzenmayer et al. , 2016). However, besides toxins, additional and so far ill-defined properties of S. aureus mediate bacterial survival after phagocytosis. Here we show that bacterial persistence in human macrophages is specific to S. aureus , whereas CoNS are less able to survive phagocytosis and are more sensitive towards low intracellular pH. There is limited information concerning replication/survival of CoNS in macrophages. The facultative pathogen S.lugdunensis (Chin et al., 2022) and S. epidermidis(Oliveira et al., 2022; Spiliopoulou et al. , 2012) were shown to be killed within macrophages. Of note, for S. epidermidis we could not link biofilm formation to bacterial survival. The biofilm positive and the biofilm negative mutant strain showed similar killing pattern within THP-1 cells. The molecular bases for the observed differences between „non-toxic“ S. aureus versus S. epidermidis remain to be elucidated but are linked to resistance towards intracellular acidification. From the available literature we could not identify any specific property that is linked to S. aureus but absent in the CoNS. Some of the S. aureus specific immune-modulatory molecules such as Eap (Stapels et al., 2014) or SPIN (de Jong et al., 2017) are likely not involved since they are strongly regulated via Sae and thus not expressed in our „non-toxic“ S. aureus strains. Conserved molecular patterns of the bacterial surface such as peptidoglycan acetylation (Bera et al., 2005) are present inS. aureus and several of the analysed CoNS. Also, the acetylation-status of lipoproteins could not be linked to the survival pattern as e.g., S. aureus and S. epidermidis show similar modification of the lipid moieties (Nguyen et al., 2017). Intracellular NOD2 signalling of peptidoglycan was linked to caspase activity, IL-1ß secretion and intracellular killing (Shimada et al. , 2010). We show that internalization of „non-toxic“ S. aureus as well as CoNS S. epidermidis resulted in IL-1ß secretion. However, the proposed inflammasome activation did obviously not result in cell-death. Inflammasomes activated caspases cleave inactive precursors of the interleukin IL-1ß and pore forming gasdermins. Cleaved gasdermin D forms transmembrane pores to enable the release of IL-1ß and also drive cell lysis through pyroptosis (Orning et al., 2019). However, cleavage of GSDMD does not uniformly lead to loss of plasma membrane integrity and cell rupture. Thus, although gasderminD is required for IL-1β secretion, this can also occur independent of cell-lysis (Evavold et al., 2018; Heilig et al., 2018).
We screened several S. aureus factors that were previously involved in protection from intracellular killing. Protection from ROS or copper might occur via synthesis of the membrane component staphyloxanthin (Liu et al. , 2008; Olivier et al. , 2009), catalase (Cosgrove, 2007 #6934}, superoxide dismutatase (Valderaset al. , 2002) or copper transporters (Purves et al. , 2018). However, mutants with deficiency in these factors were not impaired in bacterial survival in THP-1 cells. This indicates that in THP-1 cells ROS probably is not a major threat for S. aureus . However, in primary human macrophages a protective effect of SodA/M was observed. SodM is a dismutase only expressed in S. aureus . Nevertheless, expression of SodM in S. epidermidis was not sufficient to protect the bacteria from killing.
Further analysis indicates that the ability to withstand low pH is a major reason why S. aureus but not CoNS can survive within macrophages. It was previously shown that strain USA300 but not strain Newman benefit from acidification (Flannagan et al. , 2018)(Tranchemontagne et al., 2015)(Jubrail et al. , 2015; Sedlyarov et al. , 2018). We could not detect significant differences between the agr/sae negative derivatives of strain USA300 or Newman or the graRS mutant in this background. This indicates that the strain specific difference is somehow linked to Agr and/or Sae regulated factors. Both „non-toxic“ strain USA300 and Newman survive to a similar extent and are insensitive to intracellular pH. StrainS. epidermidis in contrast was protected by bafilomycin indicating that the low pH is driving intracellular killing of CoNS.