Introduction
Staphylococcus aureus asymptomatically colonizes the nose of
about 30% of the human population. Nasal carriage is a major risk
factor for severe and invasive S. aureus infections (Howden et
al., 2023; Turner et al., 2019; Wertheim et al., 2005), including
bacteremia, which occurs when this opportunistic bacterial pathogen
breaches through the epithelial barrier into the blood stream (Hommes
and Surewaard, 2022; Thwaites et al., 2011). The organism is then
rapidly phagocytosed by professional phagocytes. However, S.
aureus can withstand the killing mechanisms of professional phagocytes
and survive and replicate especially in macrophages (for reviews see
(Cole et al., 2014; Feuerstein et al., 2017; Flannagan et al., 2015;
Horn et al., 2018; Pidwill et al., 2021; Rowe et al., 2021). Through the
uptake of extracellular macromolecules macrophages deliver nutrients to
phagolysosomal S. aureus and thereby promote its growth
(Flannagan and Heinrichs, 2020). Almost all 191 analyzed clinical
isolates are internalized by macrophages and non-professional phagocytes
and a large fraction of isolates replicate and can persist within
different host cells (Rodrigues Lopes et al., 2022). However, the
intracellular life styles of individual S. aureus isolates in
non-phagocytic cells is distinct from those in macrophages indicating
different survival/killing mechanisms employed by different host cells.
Intracellular survival as well as escape from macrophages are likely
crucial for bacterial dissemination (Jorch et al., 2019; Surewaard et
al., 2016). Clinical S. aureus isolates are often deficient in
virulence gene regulators and/or in cytolytic activity (Butrico and
Cassat, 2020; Das et al., 2016; Goerke et al., 2000; Harkins et al.,
2018; Lee et al., 2021; Shopsin et al., 2008; Soong et al., 2015). Less
cytotoxic strains likely constitute a more persistent S. aureusreservoir. Thus, the genetic makeup of a given strain dictates its
capacity to either escape from cells or persist/hide for a prolonged
time (Fraunholz and Sinha, 2012; Tuchscherr et al., 2019).
There are several pathways by which intracellular bacteria are killed
within macrophages including reactive oxygen species (ROS), enzymes,
antimicrobial peptides, nutritional immunity or autophagy (Pidwillet al. , 2021). The pool of persistent bacteria in mouse
macrophages are non-growing, antibiotic resistant but metabolically
active (Peyrusson et al., 2020). Macrophage-derived ROS can promote the
formation of such persisting bacteria (Peyrusson et al., 2022; Rowe et
al., 2019), and intracellular persisters show induced expression of
several stress response pathways (Peyrusson et al. , 2020).
Acidification of the phagosome is another key mechanism involved in
killing phagocytosed bacteria. Influx of protons into the phagosome
occurs by vacuolar-type proton transporting ATPase (v-ATPase) (Lukacs et
al., 1990). S. aureusresides and multiplies in mature phagolysosomes in murine and human
macrophages (Flannagan et al. , 2015; Pidwill et al. ,
2021). Low pH promotes survival and replication of community-associated
Methicillin resistant S. aureus (caMRSA) strain USA300 (Flannagan
et al., 2018)(Tranchemontagne et al., 2015)(Sedlyarov et al., 2018).
However, this is highly strain specific and does not hold true for other
strains. E.g., compartment acidification impedes survival of strain
Newman (Jubrail et al., 2015;
Sedlyarov et al. , 2018; Tranchemontagne et al., 2015) or strain
SH1000 (Ben Shlomo et al., 2019). It was proposed that in USA300 the
intracellular activation of the two-component systems GraRS (Flannaganet al. , 2018) or Agr (Tranchemontagne et al. , 2015)
contributes to the specific adaption of this strain to the acidic
environment. Thus, whether phagosomes containing S. aureusproperly acidify and whether this leads to bacterial killing or
survival, likely depends on cell types, bacterial strains and
experimental settings (Pidwill et al. , 2021).
Coagulase-negative staphylococci (CoNS) are prototypic commensals
colonizing the human skin. However, some of the species (e.g. S.
epidermidis, S. capitis, S. lugdunensis, S. haemolyticus, S.
pettenkoferi ) are also increasingly recognised as pathogens and can
cause critical infections, especially in immunocompromised patients and
after foreign-material implantation (for reviews see (Ahmad-Mansour et
al., 2021; Argemi et al., 2019; Becker et al., 2014; Eltwisy et al.,
2022; Franca et al., 2021; Heilbronner and Foster, 2021; Heilmann et
al., 2019; Laurent and Butin, 2019; Le et al., 2018; Sabate Bresco et
al., 2017)). The fate of these species once phagocytosed is poorly
understood and to a large extent seems to be determined by the biofilm
mode of growth. E.g., biofilm-derived S. epidermidis counteract
macrophage activation (Schommer et al., 2011) and survive more
effectively in macrophages than their isogenic planktonic counterpart
(Spiliopoulou et al., 2012).
Here we compared the survival of S. aureus within human
macrophages with that of CoNS. Cytotoxic wild type S. aureus is
able to escape from macrophages through the activation of human specific
toxins hampering the analysis of bacterial persistence in these cells
(Munzenmayer et al., 2016). Therefore, we analysed non-cytotoxicagr/sae mutants which cannot escape from the cells. The
regulatory system Agr (Wang and Muir, 2016) and Sae (Liu et al., 2016)
controls the expression of most extracellular immune-modulatory factors
and toxins. Agr/sae mutants were shown to survive within the
phagolysosome for extended period without obvious harm to the host cell
(Munzenmayer et al. , 2016).
We questioned whether such
„non-toxic“ S. aureus resembles the less pathogenic CoNS species
or whether additional S. aureus specific properties account for
the intracellular survival capacity of S. aureus . We show that in
contrast to the „non-toxic“ S. aureus strains, the CoNS are
efficiently killed within 24 h post-infection in a pH dependent manner.