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.