Engineering approaches to investigate pneumococcal gene expression regulation and antibiotic resistance development

Here, we report on synthetic biology studies with a focus on the important human pathogen Streptococcus pneumoniae. First, a cloning platform is presented, which allows for the standardized assembly of genetic elements. Second, this system is used to build a selection platform, including selectable and counterselectable markers. Constitutive and inducible promoters are identified out of synthetic libraries, and gene expression circuits based on transcription repressors are constructed and characterized. These circuits include inverters, a logic AND gate and genetic toggle switches that give rise to bistable gene expression patterns. An understanding of the dynamic impact of antibiotics was key for the construction of the selection platform, and preliminary experiments suggested that growth inhibition patterns correlate with the antibiotic classification (bacteriostatic or bactericidal). These observations sparked our interest in a more detailed study of antibiotic inhibition and resistance, including observations at the single-cell level. We found that heterogeneity in antibiotic susceptibility and continued gene expression activity are important factors for resistance development. Furthermore, we found that the expression of chloramphenicol acetyltransferase in resistant cells protects susceptible cells that are present in the same environment. We show that this cross-protection establishes via intracellular antibiotic deactivation and demonstrate a potential clinical relevance of this mechanism. Finally, we present a study of the dynamics of switching behavior of multistable systems (such as virulence factor expression in the pneumococcus), on the basis of the well-characterized lac-pathway of Escherichia coli.

Dissertation: http://hdl.handle.net/11370/07eed641-f956-4e35-8061-b54b81e2cb6d