Dynamics of bacterial biofilm predation

This thesis focused on two bacterial interactions with naturally-occurring predators. The first project examined viral infection dynamics of Escherichia coli biofilms with the lytic bacteriophage T7. By combining fluorescence confocal microscopy, automated image analysis and molecular biology, this...

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Bibliographic Details
Main Author: Vidakovic, Lucia
Contributors: Drescher, Knut (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2019
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Summary:This thesis focused on two bacterial interactions with naturally-occurring predators. The first project examined viral infection dynamics of Escherichia coli biofilms with the lytic bacteriophage T7. By combining fluorescence confocal microscopy, automated image analysis and molecular biology, this investigation provided new mechanistic insights into biofilm matrix-mediated bacteriophage protection of entire bacterial communities. Bacteria infected by bacteriophages were reliably detected with the use of genetically modified T7 bacteriophages, which resulted in the production of a fluorescent protein by bacteriophage-infected bacteria. Exposure of biofilms of varying ages to T7 bacteriophages revealed a biofilm development- specific bacteriophage protection: Biofilms grown for up to 48h in microfluidic channels were susceptible to phage-mediated killing, but in contrast, biofilms that were more than 60h old survived during the constant influx of bacteriophages. Bacteriophage protection of E. coli biofilms solely depends on the presence of one component in the biofilm matrix, i.e., amyloid curli fibers. Moreover, the development of protection temporally coincides with the production of curli fibers during late stages of biofilm formation. To uncover this curli fiber-mediated protection mechanism, the biofilm architecture was analyzed and bacteriophage virions were visualized spatiotemporally in living bacterial and in vitro constructed artificial biofilms. Together, the results demonstrate that curli fibers provide a close cell- cell arrangement, filling space between the cells, so that bacteriophages cannot invade the bacterial community. In addition, curli fibers are necessary and sufficient to protect single cells from bacteriophage-mediated killing if bacteria are completely encapsulated by matrix. In conclusion, community and single-cell studies comprehensively showed how amyloid curli fibers confer collective, as well as, individual protection to biofilm-dwelling cells against bacteriophage infection. The second part of the thesis investigated the interaction of Vibrio cholerae with human macrophages (cells of the innate immune system responsible for clearance of bacteria during invasion of human tissues). V. cholerae, traditionally known to colonize the small intestine and cause the diarrheal cholera disease, was able to attach to the surface of macrophages in vitro. Once attached, bacterial biofilms were formed on the macrophage, followed by biofilm dispersal. Comprehensive examination of the interaction dynamics showed that colonization is a flagella and type IV pili-driven process. The polar flagellum, together with mannose-sensitive hemagglutinin type IV pili, facilitates bacterial attachment to the macrophage surface. During biofilm growth, V. cholerae initiates the production of toxin- coregulated type IV pili, which enhances cohesion between biofilm-dwelling bacteria and prevents complete dispersal of biofilms. Co-incubation studies revealed that production of one type of pili, mannose-sensitive hemagglutinin pili or toxin- coregulated pili, by motile V. cholerae cells is necessary and sufficient for attachment and biofilm formation. Major biofilm matrix components described for V. cholerae biofilms (RbmA, RbmC, Bap1, and VPS) are dispensable for colonization of human macrophages in vitro. These observations demonstrate an inversion of the classical predator-prey concept, by which bacteria act collectively via biofilm formation to evade phagocytosis.
Physical Description:125 Pages
DOI:https://doi.org/10.17192/z2019.0489