@phdthesis{GomezMejia2018,
  author    = {Alejandro G{\´o}mez Mejia},
  title     = {Tackling the Regulation of Pneumococcal Fitness and Virulence Factors},
  journal    = {Aufkl{\"a}rung der Regulation von Fitness- und Virulenzfaktoren in Streptococcus pneumoniae},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-22695},
  year      = {2018},
  abstract  = {Streptococcus pneumoniae is a commensal of the human upper respiratory tract and the etiological agent of several life-threatening diseases. This pathogen is the model bacterium for natural competence. Furthermore, the pneumococci played an important role in the identification of DNA as the main molecule involved in bacterial transformation. As a result, studies on the pneumococcal genome provided an initial overview of the genetic potential of this pathogen. The pneumococcus is a highly versatile bacterium possessing a high rate of uptake and recombination of exogenous DNA from neighboring bacteria. As such, a significant diversity in the genome content among the different pneumococcal strains has been reported. The capsular polysaccharide, an important pneumococcal virulence factor, is the best example on the pneumococcal diversity. There are over 98 serotypes characterized to date presenting differences in their capsule (cps) locus. Additional to the cps locus, the pneumococcus also presents 13 genomic islets annotated as regions of diversity (RD) encoded in the auxiliary genome. Remarkably, 8 of the pneumococcal RD studied so far have been associated with virulence. Furthermore, the ongoing sequencing of over 4000 pneumococcal genomes have shed light on the conservation level of well-known pneumococcal virulence factors. Interestingly, important pneumococcal virulence determinants show variations in the gene and protein sequence among the different strains. Prototypes are for example the pneumococcal surface protein C (PspC) and pneumococcal adherence and virulence factor B (PavB). Conversely, gene regulation in S. pneumoniae is carried out by highly conserved and genome- wide distributed transcriptional factors. Overall, the pneumococci interplays with its environment with 4 major regulatory systems: quorum sensing (QS), stand-alone transcriptional regulators, small RNAs (sRNAs) and two-component regulatory systems (TCS). Some of these systems are multifaceted and share more than one feature. Furthermore, there is crosstalk among the different systems, requiring the activation of a signaling cascade to function properly. A comprehensive analysis of the distribution and conservation of pneumococcal virulence factors and TCS was obtained in this study. The results are summarized as a simplified variome in which 25 pneumococcal strains with a complete sequenced genome were analyzed. Interestingly, the genes encoding the glycolytic protein enolase and the toxin pneumolysin were the most conserved virulence determinants. Additionally, the high level of conservation was confirmed for the pneumococcal TCS regulators, especially for WalKR, CiaRH and TCS08. The main focus of this study was on the regulatory functions of pneumococcal TCS. With this in mind, an extensive and detailed systematic review of the 13 pneumococcal TCS and its orphan RR was undertaken. For this purpose, every pneumococcal TCS was analyzed for its reported functional and structural information along with its contribution to the main pathophysiology of the pneumococci. In brief, S. pneumoniae can utilize its TCS for the regulation of important cellular processes and the sensing of detectable signals in the environment. Additionally, the role of TCS in pneumococcal processes and signal sensing can be divided further. In the first place, pneumococcal TCS regulate competence and fratricide, the production of bacteriocins and host-pathogen interaction processes, while the detectable signals include cell-wall perturbations, environmental stress, and nutrients. As a conclusion from this section, it is possible to analyze the pneumococcal TCS in a comprehensive manner. There is a complex network among the different pneumococcal regulators and the TCS play an important role. Moreover, these systems are highly conserved and essential for the proper functioning of the pneumococcus as a pathogen. Following up on pneumococcal TCS, this study focused especially on the TCS08. Interestingly, the pneumococcal TCS08 has been previously associated with the regulation of the cellobiose metabolism. Furthermore, this system has also been reported to regulate the expression of genes encoded in the RD4 (Pilus-1). Remarkably, the pneumococcal TCS08 was shown to be highly homologous to the SaeRS system of Staphylococcus aureus. Initially, mutant strains lacking a single (Δrr08 or Δhk08) or both components (Δtcs08) of the TCS08 were generated in pneumococcal D39 and TIGR4 strains. Transcriptomics and functional assays showed a downregulation of the PI-1 in the absence of the complete tcs08, while PavB presented an upregulation in the Δhk08 knockout. Moreover, an important number of genes coding for intermediary metabolism proteins were also found to be differentially expressed by microarray analysis. As such, the TIGR4Δhk08 strain presented a downregulation for the cellobiose operon (cel). In contrast, an upregulation was reported for the fatty acid biosynthesis (fab) and arginine catabolism (arc) operons. Conversely, a decrease in gene expression was seen in the TIGR4Δrr08 strain for the arc operon. Finally, in vivo murine pneumonia and sepsis models highlighted an involvement of TCS08 in pneumococcal virulence. Remarkably, the different TCS08 mutants presented a strain dependent effect on their virulence severity. The TIGR4Δrr08, and all TCS08 mutants in D39 showed a decrease in virulence in the pneumonia model, with no changes in sepsis. Conversely, the absence of HK08 in TIGR4 presented a highly virulent phenotype in both pneumonia and sepsis models. To sum up, the pneumococcal TCS08 influenced the expression of genes involved in fitness and colonization. Specifically, those coding for the adhesins PavB and PI-1 and fitness proteins from the cel, arc and fab operons. Remarkably, the highest changes in expression were observed in the strains lacking the HK08. Additionally, TCS08 has a strain dependent impact on pneumococcal virulence as showed by murine pneumonia and sepsis models when comparing the effects in D39 and TIGR4.},
  language  = {en}
}