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.

Interbacterial competition and anti‐predatory behavior of environmental Vibrio cholerae strains

Melanie Blokesch, Natalia Carolina Drebes Dörr

Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the hemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains’ T6SS and its actin-cross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of fourteen isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but nonidentical immunity proteins were insufficient to provide cross-immunity among those wild strains.

2020

Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae

David William Adams, Melanie Blokesch, Milena Jadwiga Jaskolska

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.

2022

Bacterial type VI secretion system facilitates niche domination

Melanie Blokesch, Natalia Carolina Drebes Dörr

Over the last few decades humanity has experienced a series of tremendous technological advances, especially in the realm of basic science. A plethora of new approaches has allowed us to appreciate life in previously unimaginable detail, prompting the realization that life in the microscopic world is not so different from what we can observe with our naked eyes. Microorganisms, for instance, like any other organism, compete with one another for resources and space. Bacteria often use simple mechanisms to occupy their niche such as rapid growth and biofilm formation. Bacteria also use ingenious strategies to maximize their success. Indeed, to engage in warfare, microorganisms often produce diffusible toxic antimicrobial compounds as well as other more complicated molecular weapons. Secretion systems are a particular kind of molecular weapon as they release or inject molecules and substrates that interact not only with hosts and predators but also with bacterial competitors. Given that the type VI secretion system (T6SS) has been recognized as a frequently used molecular weapon, it is not at all surprising that it is found widely distributed throughout diverse bacterial species (around 25% of all gram-negatives) (1). The T6SS system was discovered in Vibrio cholerae due to its toxicity against the social amoeba Dictyostelium discoideum (2). That same year, it was also suggested to contribute to the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis patients (3). Follow-up studies showed that the T6SS of these two species could also be used against other bacteria (4, 5). Indeed, while being recognized for its involvement in host–pathogen and predator–prey interactions, the principal role of the T6SS is currently thought to be as an antibacterial weapon. In PNAS, Speare et al. (6) explore an accessory T6SS of the symbiotic bacterium Vibrio fischeri and describe how this T6SS contributes to light-organ occupancy within the squid host.

2018

Bacterial type VI secretion system facilitates niche domination

Melanie Blokesch, Natalia Carolina Drebes Dörr

2018