Gene regulation in dynamic biofilms of Vibrio cholerae

The facultative human pathogen Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. Hallmarks of the lifecycle of the clinical relevant V. cholerae isolates are the transitions between two dissimilar habitats: as a natural inhabitant of the aquatic ecosystems and as a pathogen in the human gastrointestinal tracts. One key factor for environmental survival of V. cholerae is the ability to form matrix-enclosed surface-associated communities, also called biofilms. Recent studies suggest that the ability of V. cholerae to form biofilms plays not only an important role for environmental survival, but also provides an efficient way of delivering a high infectious dose to the next host. Thus, biofilm formation of V. cholerae also facilitates the transmission and spread of the disease cholera. The goal of the first activity of this study is the identification and characterization of genes induced during biofilm formation of V. cholerae. In contrast to previous approaches, we will use a reporter gene-system in combination with a hydrodynamic biofilm setup. Thus, we expect to learn more about the just recently described dynamic biofilms, which are so far uncharacterized. The presence of a dynamic flow should reflect the natural situations of V. cholerae in the aquatic environment or human host better than static conditions, which have been used previously to analyze biofilm formation. The results of this study should improve our understanding of signaling cascades, regulatory mechanisms and attachment factors important for biofilm formation as well as pathways for the biosynthesis and secretion of constituents of the biofilm matrices. Preliminary data obtained in our laboratory suggests that extracellular DNA is a novel component of the V. cholerae biofilm matrix, which will be investigated in more detail in the second activity of this research proposal. Since V. cholerae forms biofilms on chitinous surfaces in the environment and induces a state of natural competence while growing on this polymer, the presence of extracellular DNA could result in horizontal gene transfer under these conditions. A recent example for the epidemiological impact of horizontal gene transfer is the newly emerged serogroup O139, which derived from an ancestral O1 El Tor and caused devastating cholera outbreaks in the 90s. We hope that the results obtained from this study will enhance our understanding of biofilm formation of V. cholerae and perhaps other water-borne bacterial pathogens. Ideally this will accelerate the discovery and development of new therapeutic approaches targeting the persistence and transmissible forms of pathogens such as V. cholerae.

The facultative human pathogen Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. Hallmarks of the lifecycle of the clinical relevant V. cholerae isolates are the transitions between two dissimilar habitats: as a natural inhabitant of the aquatic ecosystems and as a pathogen in the human gastrointestinal tracts. One key factor for environmental survival of V. cholerae is the ability to form matrix-enclosed surface-associated communities, also called biofilms. Recent studies, including the results from this project, suggest that the ability of V. cholerae to form biofilms plays not only an important role for environmental survival, but also provides an efficient way of delivering a high infectious dose to the next host. Thus, biofilm formation of V. cholerae also facilitates the transmission and spread of the disease cholera. Using a reporter gene-system in combination with a hydrodynamic biofilm setup, we successfully identified several genes induced during biofilm formation. For example, extracellular nucleases were found to be induced during biofilm formation. Based on this observation, we identified and characterized extracellular DNA as a novel matrix component of Vibrio biofilms and important nutrient source. In addition, we could demonstrate that the nucleases also mediate the escape from the innate immune system of the host during an infection. Thus we could assign dual role of the nucleases: On the one hand during biofilm formation in aquatic environments, on the other hand during the infection in vivo. Besides several biofilm induced genes with unknown function, we identified genes involved in nutrient utilization, protein modification, signaling, motility and chemotaxis, as well as hypothetical genes. Subsequent mutagenesis of random in biofilm induced gene candidates, their phenotypic characterization and comparison under static and dynamic biofilm conditions already revealed preliminary insights into the importance of these genes in differently grown biofilms. Further comprehensive characterization of these genes will be part of the already granted follow-up FWF-project (P 27654-B16). Based on our expertise on bacterial biofilms, we were also able to assist other research groups and characterize important features of biofilms from Acinetobacter baumanii, respresenting another important human pathogen. In summary, the results obtained from this study have enhanced our understanding of biofilm formation of V. cholerae and other bacterial pathogens. Since extracellular DNA has now been found in many bacterial biofilms, the results of this project initiated new therapeutic approaches targeting this matrix component.