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.