Characterization of Genes induced during Biofilm formation of Vibrio cholerae

The severe diarrheal disease cholera is caused by the facultative human pathogen Vibrio cholerae. The life cycle of clinically relevant V. cholerae isolates is characterized by constant change between two very different ways of life: As a natural inhabitant of the aquatic environment and as pathogens in the human gastrointestinal tract. A crucial factor for survival in the environment is the formation of biofilms. Recent studies have suggested that biofilms also represent an efficient way to transmit high numbers to the next host. Therefore, biofilms of V. cholerae also contribute to the spread of the disease cholera. During the previous funding period, we have successfully identified genes, which are induced by V. cholerae during biofilm formation. Within this logical follow-up project, we will focus on the characterization of the two most interesting cascades previously identified to be biofilm induced. One represents the metabolic utilization of extracellular DNA (eDNA) as an important component in the biofilm matrix and abundant nutrient source along the life cycle of V. cholerae. So far, we could already show that two extracellular nucleases of V. cholerae degrade eDNA into nucleotides and thus can be used as a nutrient source. The aim of the present application is to characterize the necessary dephosphorylation of the nucleotides to nucleosides as well as the subsequent transport of nucleosides into the cell. Preliminary results from our laboratory suggest that V. cholerae encodes 3 nucleoside transporters with different nucleobase specificity. Unlike other bacterial nucleoside transporters all three seem to be Na+-dependent and therefore closely resemble the human representatives, which are important targets for chemotherapeutic agents. Furthermore, we want to investigate the transcriptional regulation of the enzymes involved and the physiological relevance of the metabolism of eDNA along the life cycle. In addition, our laboratory identified two O-glycosyltransferases (O-OTases) that are specifically induced in biofilms. This indicates a hitherto unknown O-glycosylation of proteins in V. cholerae. In fact, deletion of the O-OTases leads to an altered biofilm formation and morphology. Preliminary data from our laboratory suggests that extracellular proteins present in the biofilm matrix might be targets of the O-OTases. Thus, the aims of the second activity are the identification of the respective interaction partners, the transferred glycan and the regulation of O-OTases. Furthermore, the role of O-glycosylation for fitness of V. cholerae along its lifecycle will be characterized. The results of this study may improve the understanding of the physiology and biofilm formation by V. cholerae as well as other bacteria. Ideally, this will accelerate the development of new therapeutic approaches targeting the persistence and transferable types of pathogenic bacteria such as V. cholerae. Due to the high homology and functional relationship to human nucleoside transporters, the results obtained by characterization of the V. cholerae nucleoside transporters may also be used in the development of new chemotherapeutics in cancer research.

Within this research project we focused on the comprehensive characterization of the two most interesting in biofilm induced gene categories, extracellular DNA (eDNA) utilization and O-glycosylation, including their regulation cascades and association to metabolic pathways to gain a deeper insight into the complex mechanism of V. cholerae biofilm formation. In detail, utilization of eDNA as a nutrient source in the aquatic reservoirs seems to be a key factor for environmental survival, but fate of this nutrient source downstream of its degradation to nucleotides and subsequent uptake and has not been investigated so far. In silico analyses identified several periplasmic candidates for the dephosphorylation of nucleotides to nucleosides, but experimental evidence was lacking. Based on bioinformatical data we have identified three ORFs encoding nucleoside transporters and confirmed that all three are functional. Moreover, we charcterized their regulation, kinetics and specificity. Activity 1 of the research project not only comprehensively characterized nucleotide utilization in V. cholerae, but also its impact on the V. cholerae fitness at different stages of the lifecycle. Furthermore, we identified two putative O-oligosaccharyltransferases (O-OTases) of V. cholerae to be in biofilm induced. We could show that that O-glycosylation has an impact on biofilm formation in V. cholerae via interference in Type 2 secereted effectors. Since, O-glycosylation in bacteria is part of an emerging research field, we characterized the physiological role of O-glycosylation for V. cholerae as well as identify the target proteins and the transferred glycan within activity 2 of the research project.