The identification of virulence factors in E. coli biofilm formation and the role of the globin-coupled sensor EcDosC
21 June 2016
Promotiezaal (UAntwerp, Campus Drie Eiken, Building Q) - Universiteitsplein 1 - 2610 Wilrijk (Antwerp)
4:00 PM - 6:00 PM
PhD defence Joke Donné - Department of Biomedical Sciences
The pathogenicity of bacteria and their ability to form biofilms are extremely important issues because it lies at the base of many diseases of humans, animals and plants. The increase in antibiotic resistance urges the need for new alternative therapies. A promising strategy that nowadays gains more and more attention is the inhibition of bacterial virulence factors. Biofilms are forming a kind of protected environment for the bacteria to live in, with a pronounced higher tolerance to antibiotics and host immune defenses. As such, biofilms are the cause of many chronic infections. They are particularly relevant in the intensive care unit, where medical devices as central venous catheters, endotracheal tubes and urinary catheters are used on a regularly daily basis, and which are appropriate surfaces for bacterial attachment. While the diversity of microorganisms forming biofilms is quite extensive, the pathogen that mainly causes infections on medically implanted urinary catheters is Escherichia coli. The aim of this doctoral thesis was to elucidate the mechanism of E. coli biofilm formation and identify biofilm-specific virulence factors. Essential virulence factors can be targeted in alternative therapies.
First, an in vitro microtiter plate-based biofilm model for E. coli was optimized. The model allowed quantification of the total biofilm biomass with crystal violet staining and the cell viability with viable plate count. Second, a comparative SILAC-based proteomic study was performed, analyzing the differences between planktonic motile E. coli bacteria and mature sessile E. coli biofilms, to identify putative biofilm-related virulence factors. Our optimized laboratory model was used to grow and confirm the mature biofilm phenotypes. 122 proteins were found to be significantly differentially expressed. We could deduce that mature biofilm E. coli have mainly a mixed-acid fermentation metabolism to survive the low oxygen conditions within the sessile population. Moreover, the downregulated proteins in mature biofilms identified in our study are mainly involved in energy and carbohydrate metabolism and in protein synthesis, indicating a low metabolic activity of the bacteria within biofilms. In addition, several pathways which indicate their pronounced resistance characteristic are clearly upregulated in bacteria in a biofilm, such as their stress responses in order to deal with other present stress factors, and membrane synthesis.
In a more targeted approach, the role of the globin-coupled sensor EcDosC in E. coli biofilm formation was investigated. EcDosC is a c-di-GMP-synthesizing enzyme and consequently hypothesized to be a potential virulence factor since c-di-GMP is implicated in regulating bacterial motility, exopolysaccharide production and biofilm formation. Again our optimized model was used to compare the biofilm phenotypes of EcDosC overexpression and knockout E. coli strains with a wild type condition. Overexpression of EcDosC increased the biofilm mass, but not the amount of viable cells, which points to a role in the polymeric matrix production of the biofilm. Furthermore, EcDosC didn’t show an influence on the persistent property of the biofilm community, doubting the use of EcDosC as therapeutic target.
These experiments have shown that E. coli biofilms are very distinct from planktonic cultures. However, the heterogeneity of the biofilm population impedes profound research and has to be taken into account when drawing hypotheses. Future experiments should further investigate the role of all DGCs and PDEs in the process of biofilm formation. Furthermore, comparing different ages of biofilms should give a more detailed view of the development of biofilm populations.