Ontwikkeling en implementatie van tools om de cellulaire heterogeniteit in klonale populaties van Salmonella enterica te bestuderen

In this thesis genetic tools were designed and implemented to study cellular heterogeneity in clonal populations of Salmonella enterica. Salmonella is an intestinal, food-borne pathogen and was chosen as a model organism due to its large impact on human health and the global economy. The aim of this work was to develop customized transposons to study Salmonella behavior on the single cell level using time-lapse fluorescence microscopy (TLFM). Studying isogenic bacterial populations with single cell resolution can yield unprecedented insights in protein expression and function, often impossible to obtain with population level studies. The transposons constructed in this work harbor a gene encoding a fluorescent protein and after transposon mutagenesis, Salmonella mutants can be screened for unusual protein expression patterns (i.e. heterogeneous) and/or intracellular protein localization (i.e. non-diffuse localizations) using TLFM. The different transposons each serve slightly different purposes and were all validated in this work. Moreover, two major screens were set up with the developed transposons, using Salmonella Typhimurium as model organism, and atypical mutants regarding protein localization and expression could be isolated and were further characterized. Our first screen aimed for the retrieval of endogenous nucleoid reporters, which are important when studying the live impact of specific stresses on the bacterial chromosome, and uncovered the IscR protein as a valuable candidate. Although the DNA binding properties of this protein were already known, this is, to the best of our knowledge, the first time the IscR protein was studied using TLFM and further validated as a nucleoid reporter. The second screen performed in this work targeted the large virulence plasmid (pSLT) of Salmonella Typhimurium and this mobile genetic element is important for Salmonella to achieve full virulence. Different fluorescent mutants could be retrieved of which one particular mutant was found to have a bistable expression pattern, and this mutant could be linked to an important virulence operon. In depth analysis of this operon led to a more profound understanding of its regulation and more generally in how Salmonella regulates and coordinates its virulence factors. Together, these results demonstrate that our transposon mutagenesis approach, using customized transposons, is useful for screening large libraries for aberrant protein localizations and expression patterns, and can thus be the starting point in unraveling unknown protein functions and biological mechanisms in general. In turn, this will increase our knowledge regarding Salmonella behavior and may eventually lead to new approaches in combatting this pathogen.nrpages: 227status: publishe