The molecular treasure box of environmental Vibrio cholerae strains

Cholera pandemics have been affecting humankind for centuries and are still considered a major public health problem, especially in regions around the world with poor access to clean water and sanitation. Cholera pandemics are caused by a specific lineage of the bacterial pathogen Vibrio cholerae, while the vast majority of the species' diversity is found in innocuous environmental strains. The complete set of factors that allowed the emergence and success of the pandemic lineage are still unknown. Importantly, most V. cholerae strains are adapted to aquatic habitats and trigger their behavior accordingly. The aim of this thesis was therefore to gain a better understanding of what is special about the pandemic lineage when contrasted to its environmental counterparts. Specifically, we investigated environmentally important bacterial behaviors in a comparative framework. Firstly, considering the relevance of eukaryotic predation in shaping bacterioplankton structure, we investigated possible strategies used by V. cholerae to avoid grazing by the predatory amoeba Dictyostelium discoideum. We observed that V. cholerae was able to rapidly intoxicate amoebae and that the intoxication dynamics differed when caused by pandemic or environmental strains. Secondly, we compared a well-conserved set of mobile genetic elements (MGEs) of the pandemic lineage, many of which are involved in key aspects of cholera pathogenesis, with the respective counterparts of environmental strains. To do so, we sequenced and de novo assembled the genomes of fifteen environmental isolates and thoroughly described their mobilome. Moreover, transcription profiling demonstrated that most genes located on these MGEs are expressed. Thirdly, we compared environmental and pandemic strains regarding their potential to kill bacterial competitors and to defend themselves against protozoan predation. Specifically, we assessed the role of two molecular weapons: the pore-forming toxin hemolysin and the type six secretion system (T6SS), a molecular killing device that delivers effector toxins into target cells. While environmental strains keep both of these weapons constitutively active, pandemic strains employ regulatory mechanisms to control their expression. We observed that all environmental V. cholerae isolates used their T6SS to efficiently outcompete prey bacteria, while only two clades of the environmental isolates also intoxicated eukaryotic amoebae. Furthermore, we performed a meticulous in silico characterization of the effector and immunity proteins carried by the environmental strains and showed in pairwise killing experiments that a high degree of immunity protein identity was required to allow the strains' coexistence. Finally, we addressed the phenomenon of T6SS constitutive activity in non-pandemic V. cholerae strains, which has puzzled the field since the T6SS' discovery in 2006. Using a transformation-based strain library, we uncovered a genomic region that controls T6SS activity. In summary, work developed in this thesis contributes to a better understanding of V. cholerae's evolution from an innocuous inhabitant of aquatic environments to a pandemic-causing human pathogen.