@article{HungKasperkowitzKurzetal.2023, author = {Hung, Sophia and Kasperkowitz, Amelie and Kurz, Florian and Dreher, Liane and Diessner, Joachim and Ibrahim, Eslam S. and Schwarz, Stefan and Ohlsen, Knut and Hertlein, Tobias}, title = {Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1127709}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-306966}, year = {2023}, abstract = {Humanized hemato-lymphoid system mice, or humanized mice, emerged in recent years as a promising model to study the course of infection of human-adapted or human-specific pathogens. Though Staphylococcus aureus infects and colonizes a variety of species, it has nonetheless become one of the most successful human pathogens of our time with a wide armory of human-adapted virulence factors. Humanized mice showed increased vulnerability to S. aureus compared to wild type mice in a variety of clinically relevant disease models. Most of these studies employed humanized NSG (NOD-scid IL2Rgnull) mice which are widely used in the scientific community, but show poor human myeloid cell reconstitution. Since this immune cell compartment plays a decisive role in the defense of the human immune system against S. aureus, we asked whether next-generation humanized mice, like NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) with improved myeloid reconstitution, would prove to be more resistant to infection. To our surprise, we found the contrary when we infected humanized NSG-SGM3 (huSGM3) mice with S. aureus: although they had stronger human immune cell engraftment than humanized NSG mice, particularly in the myeloid compartment, they displayed even more pronounced vulnerability to S. aureus infection. HuSGM3 mice had overall higher numbers of human T cells, B cells, neutrophils and monocytes in the blood and the spleen. This was accompanied by elevated levels of pro-inflammatory human cytokines in the blood of huSGM3 mice. We further identified that the impaired survival of huSGM3 mice was not linked to higher bacterial burden nor to differences in the murine immune cell repertoire. Conversely, we could demonstrate a correlation of the rate of humanization and the severity of infection. Collectively, this study suggests a detrimental effect of the human immune system in humanized mice upon encounter with S. aureus which might help to guide future therapy approaches and analysis of virulence mechanisms.}, language = {en} } @article{HeinemannStalpBonifacioetal.2023, author = {Heinemann, Anna Sophie and Stalp, Jan Lennart and Bonifacio, Jo{\~a}o Pedro Pereira and Silva, Filo and Willers, Maike and Heckmann, Julia and Fehlhaber, Beate and V{\"o}llger, Lena and Raafat, Dina and Normann, Nicole and Klos, Andreas and Hansen, Gesine and Schmolke, Mirco and Viemann, Dorothee}, title = {Silent neonatal influenza A virus infection primes systemic antimicrobial immunity}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1072142}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304782}, year = {2023}, abstract = {Infections with influenza A viruses (IAV) cause seasonal epidemics and global pandemics. The majority of these infections remain asymptomatic, especially among children below five years of age. Importantly, this is a time, when immunological imprinting takes place. Whether early-life infections with IAV affect the development of antimicrobial immunity is unknown. Using a preclinical mouse model, we demonstrate here that silent neonatal influenza infections have a remote beneficial impact on the later control of systemic juvenile-onset and adult-onset infections with an unrelated pathogen, Staphylococcus aureus, due to improved pathogen clearance and clinical resolution. Strategic vaccination with a live attenuated IAV vaccine elicited a similar protection phenotype. Mechanistically, the IAV priming effect primarily targets antimicrobial functions of the developing innate immune system including increased antimicrobial plasma activity and enhanced phagocyte functions and antigen-presenting properties at mucosal sites. Our results suggest a long-term benefit from an exposure to IAV during the neonatal phase, which might be exploited by strategic vaccination against influenza early in life to enforce the host's resistance to later bacterial infections.}, language = {en} } @phdthesis{Ibrahim2024, author = {Ibrahim, Eslam Samir Ragab}, title = {Unraveling the function of the old yellow enzyme OfrA in \(Staphylococcus\) \(aureus\) stress response}, doi = {10.25972/OPUS-28960}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289600}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Biological systems are in dynamic interaction. Many responses reside in the core concepts of biological systems interplay (competition and cooperation). In infection situation, the competition between a bacterial system and a host is shaped by many stressors at spatial and temporal determinants. Reactive chemical species are universal stressors against all biological systems since they potentially damage the basic requirements of these systems (nucleic acids, proteins, carbohydrates, and lipids). Either produced endogenously or exogenously, reactive chemical species affect the survival of pathogens including the gram-positive Staphylococcus aureus (S. aureus). Therefore, bacteria developed strategies to overcome the toxicity of reactive species. S. aureus is a widely found opportunistic pathogen. In its niche, S. aureus is in permanent contact with surrounding microbes and host factors. Deciphering the deterministic factors in these interactions could facilitate pinpointing novel bacterial targets. Identifying the aforementioned targets is crucial to develop new strategies not only to kill the pathogenic organisms but also to enhance the normal flora to minimize the pathogenicity and virulence of potential pathogens. Moreover, targeting S. aureus stress response can be used to overcome bacterial resistance against host-derived factors. In this study, I identify a novel S. aureus stress response factor against reactive electrophilic, oxygen, and hypochlorite species to better understand its resilience as a pathogen. Although bacterial stress response is an active research field, gene function is a current bottleneck in characterizing the understudied bacterial strategies to mediate stress conditions. I aimed at understanding the function of a novel protein family integrated in many defense systems of several biological systems. In bacteria, fungi, and plants, old yellow enzymes (OYEs) are widely found. Since the first isolation of the yellow flavoprotein, OYEs are used as biocatalysts for decades to reduce activated C=C bonds in α,β-unsaturated carbonyl compounds. The promiscuity of the enzymatic catalysis is advantageous for industrial applications. However, the physiological function of OYEs, especially in bacteria, is still puzzling. Moreover, the relevance of the OYEs in infection conditions remained enigmatic.   Here, I show that there are two groups of OYEs (OYE flavin oxidoreductase, OfrA and OfrB) that are encoded in staphylococci and some firmicutes. OfrA (SAUSA300_0859) is more conserved than OfrB (SAUSA300_0322) in staphylococci and is a part of the staphylococcal core genome. A reporter system was established to report for ofrA in S. aureus background. The results showed that ofrA is induced under electrophilic, oxidative, and hypochlorite stress. OfrA protects S. aureus against quinone, methylglyoxal, hydrogen peroxide, and hypochlorite stress. Additionally, the results provide evidence that OfrA supports thiol-dependent redox homeostasis. At the host-pathogen interface, OfrA promotes S. aureus fitness in murine macrophage cell line. In whole human blood, OfrA is involved in S. aureus survival indicating a potential clinical relevance to bacteraemia. In addition, ofrA mutation affects the production of the virulence factor staphyloxanthin via the upper mevalonate pathway. In summary, decoding OfrA function and its proposed mechanism of action in S. aureus shed the light on a conserved stress response within multiple organisms.}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{Krones2022, author = {Krones, David}, title = {The Role of Acid Sphingomyelinase in \(Staphylococcus\) \(aureus\) Infection of Endothelial Cells}, doi = {10.25972/OPUS-29049}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290492}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Staphylococcus aureus is a human bacterial pathogen responsible for a variety of diseases including bacterial pneumonia and sepsis. Recent studies provided an explanation, how S. aureus and its exotoxins contribute to the degradation of endothelial junction proteins and damage lung tissue [4]. Previous findings were indicating an involvement of acid sphingomyelinase (ASM) activity in cell barrier degradation [5]. In the presented study the impact of singular virulence factors, such as staphylococcal α-toxin, on in vitro cell barrier integrity as well as their ability to elicit an activation of ASM were investigated. Experiments with bacterial supernatants performed on human endothelial cells demonstrated a rapid dissociation after treatment, whereas murine endothelial cells were rather resistant against cell barrier degradation. Furthermore, amongst all tested staphylococcal toxins it was found that only α-toxin had a significant impact on endothelial junction proteins and ASM activity. Ablation of this single toxin was sufficient to protect endothelial cells from cell barrier degradation and activation of ASM was absent. In this process it was verified, that α-toxin induces a recruitment of intracellular ASM, which is accompanied by rapid and oscillating changes in cytoplasmic Ca2+ concentration and an increased exposure of Lysosomal associated membrane protein 1 (LAMP1) on the cell surface. Recruitment of lysosomal ASM is associated, among other aspects, to plasma membrane repair and was previously described to be involved with distinct pathogens as well as other pore forming toxins (PFT). However, with these findings a novel feature for α-toxin has been revealed, indicating that the staphylococcal PFT is able to elicit a similar process to previously described plasma membrane repair mechanisms. Increased exposure and intake of surface membrane markers questioned the involvement of ASM activity in S. aureus internalization by non-professional phagocytes such as endothelial cells. By modifying ASM expression pattern as well as application of inhibitors it was possible to reduce the intracellular bacterial count. Thus, a direct connection between ASM activity and S. aureus infection mechanisms was observed, therefore this study exemplifies how S. aureus is able to exploit the host cell sphingolipid metabolism as well as benefit of it for invasion into non-professional phagocytic cells}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{Wencker2022, author = {Wencker, Freya Dorothea Ruth}, title = {The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover}, doi = {10.25972/OPUS-20712}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207124}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5'-untranslated region (5'-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes ('read-through'). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5'-region of the met mRNA. The met mRNA is degraded from its 5'-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5'-end (metI and metC) and a longer half-life towards the 3'-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon ('ON') or prevented operon transcription ('OFF'), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as 'OFF' mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum W{\"u}rzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research.}, subject = {Staphylococcus aureus}, language = {en} } @article{StelznerBoynyHertleinetal.2021, author = {Stelzner, Kathrin and Boyny, Aziza and Hertlein, Tobias and Sroka, Aneta and Moldovan, Adriana and Paprotka, Kerstin and Kessie, David and Mehling, Helene and Potempa, Jan and Ohlsen, Knut and Fraunholz, Martin J. and Rudel, Thomas}, title = {Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells}, series = {PLoS Pathogens}, volume = {17}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1009874}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-263908}, year = {2021}, abstract = {Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.}, language = {en} } @article{KronesRuehlingBeckeretal.2021, author = {Krones, David and R{\"u}hling, Marcel and Becker, Katrin Anne and Kunz, Tobias C. and Sehl, Carolin and Paprotka, Kerstin and Gulbins, Erich and Fraunholz, Martin}, title = {Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line}, series = {Frontiers in Microbiology}, volume = {12}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2021.694489}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244843}, year = {2021}, abstract = {Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.}, language = {en} } @article{MarincolaJaschkowitzKieningeretal.2021, author = {Marincola, Gabriella and Jaschkowitz, Greta and Kieninger, Ann-Katrin and Wencker, Freya D.R. and Feßler, Andrea T. and Schwarz, Stefan and Ziebuhr, Wilma}, title = {Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {11}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2021.660702}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232903}, year = {2021}, abstract = {Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.}, language = {en} } @phdthesis{Groma2021, author = {Groma, Michaela}, title = {Identification of a novel LysR-type transcriptional regulator in \(Staphylococcus\) \(aureus\)}, doi = {10.25972/OPUS-24675}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-246757}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Staphylococcus aureus is a facultative pathogen which causes a variety of infections. The treatment of staphylococcal infections is complicated because the bacteria is resistant to multiple common antibiotics. S. aureus is also known to express a variety of virulence factors which modulate the host's immune response in order to colonize and invade certain host cells, leading to the host cell's death. Among the virulence factors is a LysR-type transcriptional regulator (lttr) which is required for efficient colonization of secondary organs. In a recent report, which used transposon screening on S. aureus-infected mice, it was found that the amount of a novel lttr852 mutant bacteria recovered from the kidneys was significantly lower compared to the wildtype strains. This doctoral thesis therefore focused on phenotypical and molecular characterization of lttr852. An assessment of the S. aureus biofilm formation and the hemolysis revealed that lttr852 was not involved in the regulation of these virulence processes. RNA-sequencing for potential target genes of lttr852 identified differentially expressed genes that are involved in branched chain amino-acid biosynthesis, methionine sulfoxide reductase and copper transport, as well as a reduced transcription of genes encoding urease and of components of pyrimidine nucleotides. Promoter fusion with GFP reporters as as well as OmniLog were used to identify conditions under which the lttr852 was active. The promoter studies showed that glucose and high temperatures diminish the lttr852 promoter activity in a time-dependent manner, while micro-aerobic conditions enhanced the promoter activity. Copper was found to be a limiting factor. In addition, the impact on promoter activity of the lttr852 was tested in the presence of various regulators, but no central link to the genes involved in virulence was identified. The present work, thus, showed that lttr852, a new member of the class of LysR-type transcriptional regulators in S. aureus, has an important role in the rapid adaptation of S. aureus to the changing microenvironment of the host.}, language = {en} } @article{StelznerWinklerLiangetal.2020, author = {Stelzner, Kathrin and Winkler, Ann-Cathrin and Liang, Chunguang and Boyny, Aziza and Ade, Carsten P. and Dandekar, Thomas and Fraunholz, Martin J. and Rudel, Thomas}, title = {Intracellular Staphylococcus aureus Perturbs the Host Cell Ca\(^{2+}\) Homeostasis To Promote Cell Death}, series = {mBio}, volume = {11}, journal = {mBio}, doi = {10.1128/mBio.02250-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231448}, year = {2020}, abstract = {The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession. IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis."}, language = {en} }