@article{BlaettnerDasPaprotkaetal.2016, author = {Bl{\"a}ttner, Sebastian and Das, Sudip and Paprotka, Kerstin and Eilers, Ursula and Krischke, Markus and Kretschmer, Dorothee and Remmele, Christian W. and Dittrich, Marcus and M{\"u}ller, Tobias and Schuelein-Voelk, Christina and Hertlein, Tobias and Mueller, Martin J. and Huettel, Bruno and Reinhardt, Richard and Ohlsen, Knut and Rudel, Thomas and Fraunholz, Martin J.}, title = {Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes}, series = {PLoS Pathogens}, volume = {12}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1005857}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180380}, year = {2016}, abstract = {Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.}, language = {en} } @phdthesis{Das2018, author = {Das, Sudip}, title = {Genome-wide identification of virulence-associated genes in Staphylococcus aureus using Transposon insertion-site deep sequencing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143362}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell. This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology. Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host. Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis.}, subject = {Staphylococcus aureus}, 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} }