@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}
}
@phdthesis{GarciaBetancur2018,
  author    = {Garcia Betancur, Juan Carlos},
  title     = {Divergence of cell-fates in multicellular aggregates of \(Staphylococcus\) \(aureus\) defines acute and chronic infection cell types},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148059},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Staphylococcus aureus is a versatile human pathogen that normally develops acute or chronic infections. The broad range of diseases caused by this bacterium facilitates the escape from the host's immune response as well as from target-specific antimicrobial therapies. Nevertheless, the underlying cellular and molecular mechanisms that enable S. aureus to cause these disparate types of infections are largely unknown. In this work, we depicted a novel genetic program involved in the development of cell-fate decision, which promotes the differentiation of the staphylococcal cells into two genetically identical but differently heritable cell lines capable of defining the course of an infection, by simultaneously progressing to (i) a biofilm-associated chronic infection or (ii) a disperse acute bacteremia. Here, S. aureus growing in architecturally complex multicellular communities harbored different cell types that followed an exclusive developmental plan, resulting in a clonal heterogeneous population. We found that these cell types are physiologically specialized and that, this specialization impacts the collective behavior within the multicellular aggregates. Whereas one cell line that we named BRcells, promotes biofilm formation that engenders chronic infections, the second cell line, which we termed DRcells is planktonic and synthetizes virulence factors, such as toxins that can drive acute bacteremia. We identified that the positive feedback loop present in Agr quorum sensing system of S. aureus acts a bimodal switch able to antagonistically control the divergence of these two physiologically distinct, heritable cell lines. Also, we found that this bimodal switch was triggered in response to environmental signals particularly extracellular Mg2+, affecting the size of the subpopulations in specific colonization environments. Specifically, Mg2+-enriched environments enhanced the binding of this cation to the staphylococcal teichoic acids, increasing the rigidity of the cell wall and triggering a genetic program involving the alternative sigma factor σB that downregulated the Agr bimodal switch, favoring the enrichment of the BRcells type. Therefore, colonization environments with different Mg2+ content favored different outcomes in the bimodal system, defining distinct ratio in the BRcells/DRcells subpopulations and the S. aureus outcome in our in vitro model of development of multicellular aggregates and, the infection outcome in an in vivo mice infection model. In this prime human pathogen cell-fate decision-making generates a conserved pattern of heritable, physiological heterogeneity that actively contributes to determine the course of an infection through the emergence and spatio-temporal dynamics of distinct and specialized cell types. In conclusion, this work demonstrates that cell differentiation in pathogenic bacteria is a fundamental phenomenon and its understanding, is central to understand nosocomial infections and to designing new anti-infective strategies},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{MielichSuess2018,
  author    = {Mielich-S{\"u}ß, Benjamin},
  title     = {Elucidating structural and functional aspects of prokaryotic membrane microdomains},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162037},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Bacterial functional membrane microdomains (FMMs) are membrane platforms that resemble lipid rafts of eukaryotic cells in certain functional and structural aspects. Lipid rafts are nanometer-sized, dynamic clusters of proteins and lipids in eukaryotic cell membranes that serve as signaling hubs and assembling platforms. Yet, studying these structures can often be hampered by the complexity of a eukaryotic cell. Thus, the analogous structures of prokaryotes are an attractive model to study molecular traits of this type of membrane organization. Similar to eukaryotic lipid rafts, the bacterial FMMs are comprised of polyisoprenoid lipids, scaffold proteins and a distinct set of membrane proteins, involved in signaling or secretion. Investigating bacterial FMMs not only contributes to the understanding of the physiological importance of FMMs in bacteria, but also helps to elucidate general principles of rafts beyond prokaryotes. In this work, a bacterial model organism was used to investigate effects of synthetic overproduction of the raft scaffolding proteins on bacterial physiology. This overexpression causes an unusual stabilization of the FMM-harbored protease FtsH and therefore the proteolytic targets of FtsH are not correctly regulated. Developmental defects and aberrances in shape are the consequence, which in turn negatively affects cell physiology. These findings may be adapted to better understand lipid raft processes in humans, where flotillin upregulation is detected along with development of neurological diseases. Moreover, it was aimed at understanding the FMM-proteome of the human pathogen Staphylococcus aureus. An in-depth quantitative mass-spectrometry analysis reveals adaption of the protein cargo during different conditions, while maintaining a distinct set of core FMM proteins. As a case study, the assembly of the type VII secretion system was shown to be dependent on FMM integrity and more specifically on the activity of the FMM-scaffold flotillin. This secretion system is important for the virulence of this pathogen and its secretion efficiency can be targeted by small molecules that inhibit flotillin activity. This opens new venues for non-conventional antimicrobial compounds to treat staphylococcal infections.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}