The ability of pathogenic bacteria to cause disease depends on various virulence mechanisms. The three pathogenic species of Yersinia use a type III secretion system (T3SS) to translocate effector proteins into host cells and disrupt the immune system. This T3SS is encoded on a 70kb, low-copy, virulence plasmid. A novel mechanism of virulence was identified in Y. pseudotuberculosis, where the plasmid copy number (PCN) increases during infection. The PCN needs to be tightly regulated, as it encodes important, but costly, virulence genes. This thesis expends our understanding of PCN regulation and its importance in Yersinia virulence.

In Paper I, we demonstrate that PCN regulation as a virulence mechanism is a dynamic system capable of adapting to different host environments. We found that an increased PCN is important at the onset of infection, particularly during the colonization phase. In later stages, within different organs, the PCN decreases, suggesting a reduced need for the T3SS once the infection is established. This insight was enabled by the development of a novel method based on droplet digital PCR, allowing accurate PCN detection in sample with very little target DNA. 

In Paper II, we studied the PCN regulation by YopD. We showed that YopD represses PCN through the regulation of copA transcription. This YopD-dependant PCN control is released when YopD is secreted outside the bacteria upon contact with the host cell. YopD is a multifunctional protein. It possesses different domains crucial for its different functions. We found that the domains important for T3SS regulation are also required for PCN regulation.  

In Paper III, we used phenotypical approaches together with Nuclear Magnetic Resonance (NMR) method to study YmoA, a protein regulating gene expression in Yersinia in response to environmental stresses. YmoA’s ability to control gene expression requires its interaction with H-NS, a global DNA regulator. YmoA up-regulates a great number of genes, the T3SS and its effectors protein for instance. We observed that it also down-regulates many others, such as flagellar assembly genes. Our findings reveal that YmoA regulates PCN and senses temperature and osmotic stress resulting in a change of its conformation, which affects its ability to form a complex with H-NS. 

In summary, the studies presented in this thesis show that PCN is a highly dynamic, tightly regulated mechanism, important for Yersinia pathogenesis.