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\begin{document}

\title{Differential survival of Staphylococcal species in macrophages}



\author[1]{Christiane Wolz}%
\author[1]{Janina Bayer}%
\author[1]{Janna Becker}%
\author[1]{Xiao Liu}%
\author[1]{Lisa Gritsch}%
\author[1]{Ellen Daiber}%
\author[1]{Natalya Korn}%
\author[1]{Filipp Oesterhelt}%
\author[2]{Martin Fraunholz}%
\author[1]{Alex Weber}%
\affil[1]{Eberhard Karls Universitat Tubingen}%
\affil[2]{Julius-Maximilians-Universitat Wurzburg}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The human pathogen \emph{Staphylococcus aureus} is considered mainly an
extracellular, opportunistic pathogen, yet the bacterium is able to
survive within and escape from host cells, including macrophages. An
\emph{agr}/ \emph{sae} mutant of strain USA300 is unable to escape from
human macrophages but can replicate and survive within macrophages. We
questioned whether such ``,,non-toxic``'' \emph{S. aureus} resembles the
less pathogenic coagulase-negative Staphylococcal species (CoNS) like
\emph{S. carnosus}, \emph{S. lugdunensis}, \emph{S. capitis}, \emph{S.
warneri} or \emph{S. pettenkoferi.} We show that in contrast to the
``,,non-toxic``'' \emph{S. aureus} strains, the CoNS species are
efficiently killed within 24 h post-infection in the macrophage-like
THP-1 cells or in human primary macrophages. Bacterial persistence of
``,,non-toxic``'' S \emph{. aureus} or CoNS induced IL-1\selectlanguage{ngerman}ß release but no
cell-death. Mutations in genes coding for katalase, copprer transport or
the regulatory system GraRS or SigB did not impact bacterial survival in
THP-1 cells. Deletion of the superoxide dismutases \emph{sodA} and
\emph{sodM} impaired \emph{S. aureus} survival in human primary
macrophages but not in THP-1 cells. However, expression of the \emph{S.
aureus} specific \emph{sodM} in \emph{S. epidermidis} was not sufficient
to protect this species from being killed in THP-1 cells. Thus, at least
in those cells better bacterial survival of \emph{S. aureus} could not
be linked to higher protection from ROS. However, ``\selectlanguage{english},,non-toxic``''
\emph{S. aureus} was found to be insensitive to pH, whereas \emph{S.
epidermidis} was protected when phagosomal acidification was inhibited.
Thus, species differences seem to be linked to different sensitivity to
acidification.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

\emph{Staphylococcus aureus} asymptomatically colonizes the nose of
about 30\% of the human population. Nasal carriage is a major risk
factor for severe and invasive \emph{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, \emph{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 \emph{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 \emph{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 \emph{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 \emph{S.
aureus}reservoir. 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
(Pidwill\emph{et 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 \emph{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). \emph{S. aureus}resides and multiplies in mature
phagolysosomes in murine and human macrophages (Flannagan \emph{et al.}
, 2015; Pidwill \emph{et al.} , 2021). Low pH promotes survival and
replication of community-associated Methicillin resistant \emph{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 \emph{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
(Flannagan\emph{et al.} , 2018) or Agr (Tranchemontagne \emph{et al.} ,
2015) contributes to the specific adaption of this strain to the acidic
environment. Thus, whether phagosomes containing \emph{S.
aureus}properly acidify and whether this leads to bacterial killing or
survival, likely depends on cell types, bacterial strains and
experimental settings (Pidwill \emph{et al.} , 2021).

Coagulase-negative staphylococci (CoNS) are prototypic commensals
colonizing the human skin. However, some of the species (e.g. \emph{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 \emph{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 \emph{S. aureus} within human
macrophages with that of CoNS. Cytotoxic wild type \emph{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-cytotoxic\emph{agr/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 \emph{et al.} , 2016). We questioned whether such
,,non-toxic`` \emph{S. aureus} resembles the less pathogenic CoNS species
or whether additional \emph{S. aureus} specific properties account for
the intracellular survival capacity of \emph{S. aureus} . We show that
in contrast to the ,,non-toxic`` \emph{S. aureus} strains, the CoNS are
efficiently killed within 24 h post-infection in a pH dependent manner.



\textbf{Results}

\textbf{,,Non-toxic`` \emph{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`` \emph{agr/sae} mutant is unable to
escape after phagocytosis (M\selectlanguage{ngerman}ünzenmayer et.al, 2016). We compared
survival of wild type and \selectlanguage{english},,non-toxic`` \emph{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 \selectlanguage{english},,non-toxic`` USA300 was retained in the THP-1 cells at high
numbers. CoNS showed a significant decrease in CFU compared to the
\selectlanguage{english},,non-toxic`` USA300. Especially \emph{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
\selectlanguage{english},,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 \emph{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 \selectlanguage{english},,non-toxic``
USA300 \emph{agr/sae} mutant or the \emph{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 \emph{S.~aureus} did not
decrease. However, the number of infected macrophages harbouring
live\emph{S.~epidermidis} dropped significantly indicating that \emph{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, \selectlanguage{english},,non-toxic`` \emph{S. aureus} can survive
phagocytosis whereas a large part of the CoNS bacteria is cleared from
macrophages.

\selectlanguage{english}\textbf{,,Non-toxic`` \emph{S. aureus} strains and CoNS \emph{S.
epidermidis} induce IL1-ss after phagocytosis}

Previously, killing within macrophages was linked to inflammasome
activation (Cohen et al., 2018; Muller et al., 2015; Shimada et al.,
2010; Sokolovska et al., 2013). We questioned whether
\selectlanguage{english},,non-toxic``\emph{S. aureus} was still able to provoke IL-1ss release as
read-out for inflammasome activation. Since there are also major
differences between\emph{S. aureus} strains (Pidwill \emph{et al.} ,
2021), we included an\emph{agr/sae} mutant of strain Newman into the
analysis. There was no significant difference between bacterial survival
or cytotoxicity of \selectlanguage{english},,non-toxic`` USA300 versus \selectlanguage{english},,non-toxic`` strain Newman
(Fig. 2A, 2B). Interestingly, IL-1ss was detectable not only in cells
infected with the \selectlanguage{english},,non-toxic`` \emph{S. aureus} strains but also in
those infected with\emph{S.~epidermidis} (Fig. 2C). IL-1ss release
indicates that intracellular \emph{S. aureus} as well as CoNS induce
inflammasome activation. However, since there is no significant
difference between species, inflammasome activation doesn't account for
the differences observed in bacterial survival. We next tested bacterial
survival, cytotoxicity and IL-1ss release in human primary macrophages
(hMDM). Again CoNS \emph{S. epidermidis} was more efficiently killed
compared to \selectlanguage{english},,non-toxic`` \emph{S.~aureus} (Fig. 2D). IL-ss release was
not significantly different between species (Fig. 2F).

\textbf{Role of ROS detoxifying enzymes for bacterial survival}

Killing of phagocytosed bacteria might occur via different mechanisms.
Several \emph{S. aureus} factors were shown to contribute to protection
against intoxication by copper ((\emph{copXL, copA} ) (Purves et al.,
2018)), ROS (staphyloxanthin biosynthesis (Liu et al., 2008; Olivier et
al., 2009)) or H\textsubscript{2}0\textsubscript{2} (\emph{katA} ,
(Cosgrove et al., 2007)). Inactivation of \emph{copA} , \emph{copL} ,
or\emph{katA} in the \selectlanguage{english},,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 \emph{sigB} mutants non-pigmented. Since deletion
of \emph{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 \emph{et al.} , 2018). However, in
the\emph{agr/sae} negative background no significant difference in
intracellular survival was observed (Fig. 3A)

Interestingly, \emph{S. aureus} possesses two \emph{sod}
genes,\emph{sodA} and \emph{sodM} , which is unique among Gram-positive
bacteria (Valderas et al., 2002). Other staphylococci only possess one
Sod homodimer resembling \emph{S. aureus} SodA. Thus, we speculated that
\emph{sodM} may contribute to the unique survival of \emph{S. aureus} in
macrophages. But when we deleted\emph{sodA} , \emph{sodM} or both genes
in the \selectlanguage{english},,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 \emph{sodA-sodM} -mutant
(Figure 3E). We next overexpressed\emph{sodM} in \emph{S. epidermidis}
using an anhydro-tetracycline inducible promoter. SodM expression could
not rescue \emph{S. epidermidis} . Thus, the mutant analyses could not
link the better survival of \emph{S. aureus} versus CoNS in THP-1 cells
to any property to deal with ROS or toxic copper. In hMDMs \emph{S.
aureus} specific expression of two superoxide dismutases contributes to
bacterial survival but cannot protect \emph{S. epidermidis} from being
killed.

\selectlanguage{english}\textbf{Intracellular acidification is linked to killing of CoNS but not
of ,,non-toxic``\emph{S. aureus}}

\emph{S. aureus} resides and multiplies in mature phagolysosomes in
murine and human macrophages (Flannagan \emph{et al.} , 2015;
Pidwill\emph{et al.} , 2021). Low pH even promotes survival of USA300
whereas other \emph{S. aureus} strains such as strain Newman seem to be
sensitive to low pH (Flannagan \emph{et al.} , 2018)(Tranchemontagne et
al., 2015)(Sedlyarov \emph{et al.} , 2018). We assayed whether
\selectlanguage{english},,non-toxic`` \emph{S. aureus} or CoNS strains were sensitive to the
v-ATPase inhibitor bafilomycin, which inhibits lysosomal acidification
(Sedlyarov \emph{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 \selectlanguage{english},,non-toxic`` \emph{S. aureus} strains USA300 and
Newman are insensitive towards pH alterations in the phagosomes.
Survival of CoNS \emph{S. epidermidis} was significantly increased in
bafilomycin treated cells. However, \emph{S. epidermidis} was not found
to be more sensitive to pH when grown in medium (Fig. S2).

\textbf{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 \emph{S. aureus} (Hommes and Surewaard, 2022;
Pidwill\emph{et al.} , 2021). \emph{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 \emph{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 \emph{et al.} , 2016). However, besides
toxins, additional and so far ill-defined properties of \emph{S. aureus}
mediate bacterial survival after phagocytosis. Here we show that
bacterial persistence in human macrophages is specific to \emph{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.\emph{lugdunensis} (Chin et al., 2022) and
\emph{S.~epidermidis}(Oliveira et al., 2022; Spiliopoulou \emph{et al.}
, 2012) were shown to be killed within macrophages. Of note, for
\emph{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 \selectlanguage{english},,non-toxic`` \emph{S. aureus}
versus \emph{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
\emph{S. aureus} but absent in the CoNS. Some of the \emph{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 \selectlanguage{english},,non-toxic``
\emph{S. aureus} strains. Conserved molecular patterns of the bacterial
surface such as peptidoglycan acetylation (Bera et al., 2005) are
present in\emph{S. aureus} and several of the analysed CoNS. Also, the
acetylation-status of lipoproteins could not be linked to the survival
pattern as e.g., \emph{S. aureus} and \emph{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 \emph{et al.} , 2010). We
show that internalization of \selectlanguage{english},,non-toxic`` \emph{S. aureus} as well as
CoNS \emph{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-1\selectlanguage{greek}β \selectlanguage{english}secretion,
this can also occur independent of cell-lysis (Evavold et al., 2018;
Heilig et al., 2018).

We screened several \emph{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 \emph{et al.} , 2008; Olivier \emph{et al.} ,
2009), catalase (Cosgrove, 2007 \#6934\}, superoxide dismutatase
(Valderas\emph{et al.} , 2002) or copper transporters (Purves \emph{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 \emph{S.~aureus} .
However, in primary human macrophages a protective effect of SodA/M was
observed. SodM is a dismutase only expressed in \emph{S.~aureus} .
Nevertheless, expression of SodM in \emph{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 \emph{S.~aureus} but not CoNS can survive within
macrophages. It was previously shown that strain USA300 but not strain
Newman benefit from acidification (Flannagan \emph{et al.} ,
2018)(Tranchemontagne et al., 2015)(Jubrail \emph{et al.} , 2015;
Sedlyarov \emph{et al.} , 2018). We could not detect significant
differences between the \emph{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. Strain\emph{S.
epidermidis} in contrast was protected by bafilomycin indicating that
the low pH is driving intracellular killing of CoNS.

\textbf{Methods}

\textbf{Strains and growth conditions}

Staphylococcal strains used in this work are listed in Supp. Table S1.
All strains were grown in Tryptic Soy Broth, 37\selectlanguage{ngerman}°C, 180 rpm. For strains
carrying resistance genes, antibiotics (10 \selectlanguage{greek}µ\selectlanguage{english}g/ml) used only in
precultures. Bacteria from an overnight culture were diluted to an
initial optical density OD\textsubscript{600} = 0.2 and grown for 2 h.
For \emph{S. epidermidis} strain carrying an AHT-inducible plasmid
(pCG817), AHT-induction (0.1~\selectlanguage{greek}µ\selectlanguage{english}g/ml) was started in day cultures and
maintained during the whole experiment until t0, t3 or t24.

To check if bacterial growth is not decreased by bafilomycinA1
treatment, bacteria from overnight culture were diluted to an initial
optical density OD\textsubscript{600} = 0.05 in a 96-flat-bottom-plate
with or without BafilomycinA1 (0.1 \selectlanguage{greek}µ\selectlanguage{english}M). Absorbance were measured with a
wavelength of 600 nm for 12 h in a Tecan-reader.

For pH dependent growth, bacteria from an overnight culture were diluted
to an initial optical density OD\textsubscript{600} = 0.05 in LB-medium
with pH7 or pH5 in a 96-flat-bottom-plate (Greiner). Absorbance were
measured with a wavelength of 600 nm for 12 h in a Tecan-reader.

\textbf{Strain construction}

All derivatives of the USA300 \emph{agr/sae} mutants were obtained by
transduction of specific mutations from the Nebraska mutant library (Fey
et al., 2013) into strain USA300 \emph{agr/sae} using \selectlanguage{greek}Φ\selectlanguage{english}11 lysates. All
mutants were confirmed by PCR using oligonucleotides spanning the
transposon insertion sites. For expression of sodM
in\emph{S.~epidermidis} , sodM was amplified using
oligonucleotides~5'-CCAGTGAATTCGAGCTCAAATTATATTAAGTTATATTATTTTGCTGCTTGGT-3
and 5'-ATGATGGTACCGTTAACAACACACCCGAAATTAATTATT-3. The construct was
cloned into the AHT-inducible vector pCG248. The generated plasmid
pCG817 was transduced into \emph{S. epidermidis} 1457 using \emph{S.
aureus} PS187 \selectlanguage{greek}Δ\selectlanguage{english}\emph{sau} \selectlanguage{greek}Δ\selectlanguage{english}\emph{hsdR} and the phage
\selectlanguage{greek}Φ\selectlanguage{english}187\emph{.}Transductants~were~verified~with~5'-CAAAATTATACATGTCAACGA-3'~and
5'-AAGCAGCTCTAATGCGCTGT-3'

\textbf{Cell culture}

THP-1-CWT cells containing the recruitment marker YFP-CWT (recognizing
Gram-positive peptidoglycan) (Grosz et al., 2013) were grown in RPMI
1640 medium (Biochrom) with 2mM glutamine, 10\% heat-inactivated foetal
bovine serum (Sigma), 2\% HEPES (Biochrom), 1\% penicillin/streptomycin
(Gibco) and 1mM sodium pyruvate (SigmaAldrich). Cell viability was
determined by trypan blue staining and was at least 90\% before all
experiments. To induce differentiation, 1 \selectlanguage{ngerman}×
10\textsuperscript{6}cells/ml were treated with 160nM
phorbol-12-myristate-13-acetate (PMA) for 48 h. After differentiation,
the cells became adherent to the culture dishes. The day of infection
differentiated cells were washed twice with Hank's Balanced Salt
Solution (HBSS) and further incubated with RPMI medium with 10\%
heat-inactivated fetal bovine serum and 10\% human Serum (Sigma) until
infection.

\textbf{Preparation of human monocyte-derived macrophages}

Monocytes were isolated from the peripheral blood by Ficoll/Histopaque
gradient centrifugation. Cells were washed once in phosphate-buffered
saline (PBS) and adjusted to a cell of 2 × 10\^{}6 ml in RPMI 1640
medium (Biochrom) with 2mM glutamine, 10\% heat-inactivated fetal calf
serum (FCS, Sigma), 2\% HEPES (Biochrom), 1\% penicillin/streptomycin
(Gibco) and 1mM sodium pyruvate (Sigma-Aldrich). 500\selectlanguage{greek}µ\selectlanguage{english}l was seeded into
each well of 24- well tissue culture. After 1 h of incubation, cells
were washed twice with PBS to remove non-adherent cells. Cells were
further incubated with medium containing 25 nM granulocyte macrophage
colony stimulating factor (PeproTech), which was additionally added
every second day. The cells were further polarized into M1 macrophages
with lipopolysaccharide (LPS, 100 ng/\selectlanguage{greek}μ\selectlanguage{english}l) one day before start of the
experiment. After 7 days of incubation, cells were used for experiments.

\textbf{Intracellular survival assay}

The assays were performed in a 24-well tissue culture plates (500\selectlanguage{greek}µ\selectlanguage{english}l
THP-1-CWT cells per well). Bacterial cultures were washed twice with
sterile PBS and adjusted to reach a multiplicity of infection of 10:1.
Cells were infected and incubation time for phagocytosis was 1 h. The
cells were then washed once with HBSS, and the remaining extracellular
bacteria were killed by incubation with lysostaphin (2 \selectlanguage{greek}μ\selectlanguage{english}g/ml) and
gentamicin (200 \selectlanguage{greek}μ\selectlanguage{english}g/ml). After 1 h (t0) the cells were washed twice with
HBSS and then incubated in HBSS containing gentamicin (200 \selectlanguage{greek}μ\selectlanguage{english}g/ml) for 24
h (t24). At the indicated time points, the cells were washed once with
HBSS and incubated in 0.1\% TritonX-100 for 5 min to disrupt the host
cells. Appropriate dilutions were plated on tryptic soy agar plates and
incubated at 37\selectlanguage{ngerman}°C for the enumeration of colony forming units (CFU) on
the following day. The Inhibitor for acidification of the phagosome,
BafilomycinA1 (0.1 \selectlanguage{greek}µ\selectlanguage{english}M) (Sigma) was added after 1 h of phagocytosis until
indicated timepoints.

\textbf{Cytotoxicity assay (LDH)}

Lactate dehydrogenase (LDH) activity was used as an index of
cytotoxicity. At indicated time points 100\selectlanguage{greek}µ\selectlanguage{english}l supernatant of infected
cells (intracellular survival assay) were collected and stored at 4\selectlanguage{ngerman}°C
until assay was performed. 1\% Triton on cells were used as a positive
control. The Cytotoxicity Detection kit (Roche Diagnostics GmbH) was
used like manufacturer's instructions and activity was determined with
an adsorption of 440 nm. Values of the measurement after 15 min were
used for calculation of the percentage of cytotoxicity with the 1\%
TritonX-100 positive control as 100\%.

\textbf{Live-/Dead staining}

THP-1-CWT cells were seeded with 300~\selectlanguage{greek}µ\selectlanguage{english}l/well of a
1[?]10\textsuperscript{6~}cells/ml cell suspension in THP-1 culture medium
on a microscopy IBIDI-slide and stimulated with 160~nM Phorbol
12-myristate 13-acetate (PMA). Infection was done like already described
with an MOI of 10. At the respective timepoint, the cells were washed
with HBSS and then fixed with 150~\selectlanguage{greek}µ\selectlanguage{english}l ice-cold Fix Mix for 30 min at RT.
Cells were permeabilised with 300~\selectlanguage{greek}µ\selectlanguage{english}l 0.1~\% TritonX-100 in PBS for 5 min
(RT) and then stained with 150~\selectlanguage{greek}µ\selectlanguage{english}l staining solution for 15 min at RT in
the dark. The staining solution was prepared with 1.5~\selectlanguage{greek}µ\selectlanguage{english}l of each dye
component A and B per~ml~PBS (LIVE/DEAD BacLight. Bacterial Viability
Kits, Invitrogen). The cells were finally washed three times with HBSS
and covered with three drops of Dako fluorescence mounting medium
(Agilent Technologies). The slides were stored at 4~\selectlanguage{ngerman}°C in the dark until
imaging. Microscopy was performed with a LSM800 microscope (Zeiss) using
a 63~x objective with immersion oil. The propidium iodide (PI) channel
received an excitation wavelength of 305~nm, an emission wavelength of
617~nm and a detection wavelength of 576-700~nm. For detection of Syto9,
an excitation wavelength of 483~nm, an emission wavelength of 500~nm and
a detection wavelength of 495-560~nm were used.

\textbf{Live-Imaging}

For Live-cell Microscopy cells were infected with bacteria
constitutively expressing mCherry. THP1-CWT cells were seeded in a 4
Compartment Cell Culture Dish (Greiner) and infection was performed like
already described. Live-Imaging was started at t0 with a LSM800
microscope (Zeiss) using a 40 x objective with immersion oil. The yellow
fluorescent protein (YFP) channel received an excitation wavelength of
488~nm, an emission wavelength of 509~nm and a detection wavelength of
490-575~nm. For detection of mCherry, an excitation wavelength of
587~nm, an emission wavelength of 610~nm and a detection wavelength of
565-700~nm were used.

\textbf{ELISA}

100\selectlanguage{greek}µ\selectlanguage{english}l supernatant was collected from escape-assay and stored at -20\selectlanguage{ngerman}°C
until used. ELISA was performed after the instruction of the producer.
This was performed by the ELISA MAX Deluxe Set Human IL-1ß kit
(BioLegend).

\textbf{Statistical analysis}

Statistical analyses were performed using GraphPad Prism software.
Significance was determined by one-way analysis of variance with Tukey`s
multiple comparison post-test. For comparison of two groups significance
was determined by unpaired t-test. * P\textless{}0.1; **
P\textless{}0.01; ***\textless{}0.001; ****\textless{}0.0001.

\textbf{Ethics}

Blood was taken from healthy donors which provided their written
informed consent before participation. Approval for use of biomaterials
was obtained for this project by the Medical Faculty Tübingen in
accordance with the principles laid down in the Declaration of Helsinki
as well as applicable laws and regulations.

\textbf{Acknowledgement}

This work was funded by Deutsche Forschungsgemeinschaft: Transregio156,
(Project 246807620, CW) and Schwerpunktprogramm Spp2225 to CW and AW
(Project 446507619). Infrastructural funding was provided by the
Deutsche Forschungsgemeinschaft (DFG), Cluster of Excellence EXC 2124
``Controlling Microbes to Fight Infections'' (Project 390838134). We
thank Libera Lo Presti for scientific discussions and editing of the
manuscript and Bernhard Krismer for providing CoNS isolates.

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\textbf{Legend to the Figures:}

\textbf{Figure 1: ``Non-toxic'' \emph{S. aureus} survive better in
macrophages than CoNS.}

After phagocytosis and lysostaphin/gentamicin treatment for 1 h (t0)
THP-1-CWT cells were further incubated for 24 h in medium containing
gentamicin to kill extracellular/escaped bacteria. At indicated time
points, the cells were lysed and bacterial CFU determined (\textbf{A} ).
Membrane integrity of the THP-1- CWT cells were assessed by LDH-assay
(\textbf{B} ).

Bacteria containing macrophages were enumerated by live/death staining.
Cells were seeded in an IBIDI-slide (\textbf{C} ). Quantification of
Syto9 positive macrophages (\textbf{D} ). Quantification of Syto9
positive bacteria in positive macrophages (\textbf{E} ).

The data represent at least three biological replicates. Significance
was determined by one-way analysis of variance with Tukey`s multiple
comparison post-test (panel \textbf{A, B, D} ) or students t-test for
panel \textbf{E} .

\textbf{Figure 2: ``Non-toxic'' \emph{S. aureus} strains and
CoNS\emph{S. epidermidis} induce IL1-ß after phagocytosis}

After phagocytosis and lysostaphin/gentamicin treatment for 1 h (t0)
THP-1-CWT cells (\textbf{A,~B,~C} ) or hMDM (\textbf{D, E, F} ) were
further incubated for 3 h or 24 h in medium containing gentamicin to
kill extracellular/escaped bacteria. At indicated time points, the cells
were lysed and bacterial CFU determined. Intracellular bacteria are
expressed as the ratio of CFU at t3 or t24 to CFU at t0 (\textbf{A, D}
). Membrane integrity of the THP-1- CWT cells were assessed by LDH-assay
(\textbf{B, E} ) and IL-1ß by ELISA (\textbf{C, F} ).

The data represent at least three biological replicates. Significance
was determined by one-way analysis of variance with Tukey`s multiple
comparison post-test.

\textbf{Figure 3: Role of ROS detoxifying enzymes and regulatory system
for bacterial survival.}

After phagocytosis and lysostaphin/gentamicin treatment for 1 h (t0)
THP-1-CWT cells (\textbf{A, B, C, D} ) or hMDM (\textbf{E, F} ) were
further incubated for 3 h or 24 h in medium containing gentamicin to
kill extracellular/escaped bacteria. At indicated time points, the cells
were lysed and bacterial CFU determined (\textbf{A, C, E} ). Membrane
integrity of the THP-1-CWT or hMDM cells were assessed by LDH-assay
(\textbf{B, D, F} ). The data represent at least three biological
replicates Significance was determined by one-way analysis of variance
with Tukey`s multiple comparison post-test.

\textbf{Figure 4: Intracellular acidification is linked to killing of
CoNS but not non-toxic \emph{S.~aureus}.}

After phagocytosis and lysostaphin/gentamicin treatment for 1 h (t0)
THP-1-CWT cells were further incubated for 3 h or 24 h in medium
containing gentamicin to kill extracellular/escaped bacteria. At
indicated time points, the cells were lysed and bacterial CFU determined
(\textbf{A} ). Membrane integrity of the THP-1, CWT cells were assessed
by LDH-assay (\textbf{B} ).

The data represent at least three biological replicates. Significance
was determined by one-way analysis of variance with Tukey`s multiple
comparison post-test.

\textbf{Supplements}

\textbf{Table S1: strains and plasmids}

\textbf{Movies S1-4:} Live-cell microscopy of THP-1-CWT cells infected
with constitutively expressing mCherry. Uninfected control (S1), USA300
wild type (S2), USA300 agr/sae (S3), S.~epidermidis (S4)

\textbf{Figure S1: Bacterial growth is not decreased by bafilomycinA1
treatment}

\textbf{Figure S2: Bacterial growth is not decreased by an acidic
environment.}\selectlanguage{english}
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