@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} } @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} } @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{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} } @article{GromaHorstDasetal.2020, author = {Groma, Michaela and Horst, Sarah A. and Das, Sudip and Huettel, Bruno and Klepsch, Maximilian and Rudel, Thomas and Medina, Eva and Fraunholz, Martin}, title = {Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection}, series = {mbio}, volume = {11}, journal = {mbio}, number = {4}, doi = {10.1128/mBio.01646-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230473}, year = {2020}, abstract = {Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment. IMPORTANCE Staphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.}, language = {en} } @article{DrayssClausHubertetal.2019, author = {Drayß, Maria and Claus, Heike and Hubert, Kerstin and Thiel, Katrin and Berger, Anja and Sing, Andreas and van der Linden, Mark and Vogel, Ulrich and L{\^a}m, Thi{\^e}n-Tr{\´i}}, title = {Asymptomatic carriage of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Group A Streptococcus and Staphylococcus aureus among adults aged 65 years and older}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0212052}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201042}, pages = {e0212052}, year = {2019}, abstract = {Objective The aim of this study was to determine the prevalence of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, group A Streptococcus (GAS), and Staphylococcus aureus in asymptomatic elderly people and to unravel risk factors leading to colonization. Methods A multi-centre cross-sectional study was conducted including 677 asymptomatic adults aged 65 years or more, living at home or in nursing homes. Study areas were Greater Aachen (North-Rhine-Westphalia) and Wuerzburg (Bavaria), both regions with medium to high population density. Nasal and oropharyngeal swabs as well as questionnaires were collected from October 2012 to May 2013. Statistical analysis included multiple logistic regression models. Results The carriage rate was 1.9\% ([95\%CI: 1.0-3.3\%]; 13/677) for H. influenzae, 0.3\% ([95\%CI: 0-1.1\%]; 2/677) for N. meningitidis and 0\% ([95\% CI: 0-0.5\%]; 0/677) for S. pneumoniae and GAS. Staphylococcus aureus was harboured by 28.5\% of the individuals ([95\% CI: 25.1-32.1\%]; 193/677) and 0.7\% ([95\% CI: 0.2-1.7\%]; 5/677) were positive for methicillin-resistant S. aureus. Among elderly community-dwellers colonization with S. aureus was significantly associated with higher educational level (adjusted OR: 1.905 [95\% CI: 1.248-2.908]; p = 0.003). Among nursing home residents colonization was associated with being married (adjusted OR: 3.367 [1.502-7.546]; p = 0.003). Conclusion The prevalence of N. meningitidis, H. influenzae, S. pneumoniae and GAS was low among older people in Germany. The S. aureus rate was expectedly high, while MRSA was found in less than 1\% of the individuals.}, language = {en} } @article{GarciaBetancurGoniMorenoHorgeretal.2017, author = {Garc{\´i}a-Betancur, Juan-Carlos and Go{\~n}i-Moreno, Angel and Horger, Thomas and Schott, Melanie and Sharan, Malvika and Eikmeier, Julian and Wohlmuth, Barbara and Zernecke, Alma and Ohlsen, Knut and Kuttler, Christina and Lopez, Daniel}, title = {Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus}, series = {eLife}, volume = {6}, journal = {eLife}, number = {e28023}, doi = {10.7554/eLife.28023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170346}, year = {2017}, abstract = {A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.}, language = {en} } @article{MielichSuessWagnerMietrachetal.2017, author = {Mielich-S{\"u}ss, Benjamin and Wagner, Rabea M. and Mietrach, Nicole and Hertlein, Tobias and Marincola, Gabriella and Ohlsen, Knut and Geibel, Sebastian and Lopez, Daniel}, title = {Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {11}, doi = {10.1371/journal.ppat.1006728}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170035}, pages = {e1006728}, year = {2017}, abstract = {Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.}, language = {en} }