Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae

Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements—in particular bacteriophages and plasmids—the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2–4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shaped the evolution of the most successful lineage of pandemic V. cholerae.

The molecular treasure box of environmental Vibrio cholerae strains

Natalia Carolina Drebes Dörr

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.

EPFL2021

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

Horizontal gene transfer (HGT) has a major impact on bacterial evolution, leading to acquisition or deletion of genes and gene clusters, including those encoding antibiotic resistances and virulence factors. HGT therefore contributes to pathogen emergence, which can have a major impact on human health. As a mode of HGT, natural competence for transformation allows bacteria to directly acquire DNA from the environment and to integrate parts of this genetic material within their genomes. The human pathogen Vibrio cholerae is naturally competent when it grows in association with the chitinous exoskeletons of zooplankton. Under this condition, the competence state is initiated by the regulatory protein TfoX, which co-induces a molecular killing device known as the type VI secretion system (T6SS). The co-regulation of the T6SS and competence results in the lysis of non-immune neighboring bacteria and the subsequent acquisition of their DNA, which fosters HGT. In V. cholerae, the T6SS is also activated by the TfoX homologue protein, named TfoY. In this study, we aimed at studying the regulatory pathways and the co-regulation of interbacterial predation and DNA uptake in order to provide new insights into the environmental lifestyle of V. cholerae and other vibrios. We first investigated the conserved function of TfoX- and TfoY in V. fischeri, V. alginolyticus, and V. parahaemolyticus using diverse methods such as qRT-PCR, interbacterial killing assays, and motility assessments. We demonstrated that TfoX-mediated T6SS and competence induction and TfoY-induced motility are conserved phenotypes in the tested Vibrio species, whereas the TfoY-mediated T6SS regulation varied. These variations might reflect diverse defense mechanisms, as these different species have adapted to cope with their environmental niches. Next, we tested the outcome of the TfoX-mediated co-regulation of the T6SS and competence in V. cholerae. Under conditions mimicking the bacteriumâs natural habitat, we showed for the first time the extent of DNA that was integrated on V. choleraeâs genome after T6SS-mediated interbacterial predation. Indeed, we demonstrated that T6SS-dependent bacterial predation not only increased the transformation rates but also fostered the exchange of multiple and huge fragments of DNA. We also showed the necessity of an exquisite co-regulation between the T6SS, the competence-related DNA uptake machinery, and the competence-repressed nuclease. Finally, we disclosed that prey-released DNA is not a private good of the attacking bacterium but also accessible to other surrounding cells that have likewise entered the competence state. Altogether, our findings shed light on the conservation of the TfoX- and TfoY-driven phenotypes in several Vibrio species. We also contributed to a better understanding of the interplay between neighbor predation and DNA uptake and the consequences that this interplay has on genome plasticity.

EPFL2019