Dangerous acquaintances: the interplay between type IV pili and the type VI secretion system during Vibrio cholerae's environmental lifestyle

Cholera, caused by the bacterium Vibrio cholerae, has affected humanity throughout history and still impacts millions of people every year. Apart from being a human pathogen, V. cholerae is a common member of the aquatic environment. Due to this natural reservoir, seasonal outbreaks of cholera occur in endemic regions of the world. Such outbreaks are frequently associated with estuarine floodings and zooplankton blooms. The chitinous exoskeletons of zooplankton serve as a nutrient source for V. cholerae and induce an interbacterial killing device known as the type VI secretion system (T6SS) and a type IV pilus (T4P) structure known as the DNA-uptake pilus. These molecular nanomachines are involved in interbacterial competition, adherence to chitin, and kin-specific aggregation, respectively. The latter phenotype relies on the interaction of pili composed of identical major pilin subunits. It is important to note that the encoding allele varies between different strains of V. cholerae. How these co-regulated systems contribute to the success of V. cholerae in the environment remains elusive. The aim of this thesis was therefore to understand the interplay between T6SS and T4P in order to explore how these systems assist V. cholerae in overcoming challenges within its environment. First, we attempted to reconstitute and study the DNA-uptake pilus in E. coli. Unfortunately, heterologous expression of the DNA-uptake assembly platform was toxic to the host cell and did not allow functional testing of the pilus in E. coli. Next, we tested the aggregative phenotype of the pilus in mixed V. cholerae cultures. Here, we showed that T4P can facilitate T6SS-mediated killing under liquid conditions in a major pilin-specific manner. The ability of T4P to facilitate T6SS predation was conserved among major pilin variants capable of aggregation. Notably, we identified "promiscuous" T4P that, despite their differences, facilitated interactions, enabling T6SS-mediated competition. Importantly, even in such cases, there was a preference for interaction with kin, narrowing T6SS competition interfaces to a subset of the population. This phenomenon resulted in the formation of dead cell barriers. We therefore concluded that the natural diversity of T4P dictates the extent of T6SS competition and may function as a defence strategy of V. cholerae against T6SS invaders. Finally, our aim was to investigate the role of T4P and T6SS when V. cholerae grows on biotic surfaces. To do so, we worked on developing a protocol that mimics a chitin surface in the natural aquatic environment. Initial data suggests T4P-dependent T6SS killing of sensitive strains. However, in our current protocol colonisation onto the chitin surface is not dependent on the DNA-uptake pilus. In summary, this work contributes to a better understanding of the interplay between T4P and T6SS during the environmental lifestyle of V. cholerae.