Investigating the role and dynamics of type IVa pili in a predatory bacterium

Details

Supervisors

Laloux, Géraldine ; Hallet, Bernard

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

enPredation is a fundamental ecological interaction, including within bacterial communities. Some bacteria have developed sophisticated strategies to actively kill other bacteria and feed on their cellular contents. This is notably the case of Bdellovibrio bacteriovorus, an obligate predatory bacterium that relies exclusively on prey consumption for its growth and survival. This predator employs a remarkable predation strategy whereby it attaches to its prey, invades its periplasm, and then digests it from within to fuel its own growth, culminating in the production of numerous daughter cells. Although the molecular mechanisms underlying this lifestyle remain largely unknown, preliminary observations suggest that B. bacteriovorus employs different macromolecular systems to mediate its interaction with prey. Among these are pili, notably a type IVa pilus (T4aP), dynamically polymerized by a macromolecular machinery that spans the bacterial envelope. This system could be involved in several critical steps of prey interaction, particularly during prey recognition, attachment, or periplasmic invasion. This study therefore aims to characterize this system to enhance our understanding of the molecular mechanisms at play during the interaction between B. bacteriovorus and its prey. Building on bioinformatic analyses of the genome previously performed by a collaborator, I first proceeded to identify the genes encoding the T4aP system in this model. The identified components were subsequently investigated in silico by sequence analysis to determine their architecture. In parallel, a transcriptomic data analysis established that the genes encoding T4aP display an expression profile consistent with a role in the early phases of predation. In a second phase, I constructed translational fusions between T4aP components and fluorescent proteins to track their subcellular dynamics throughout the predator's cell cycle. Microscopic observation shows that T4aP is localized at the so-called "invasive" cell pole – the pole through which the predator penetrates its prey. This unipolar localization, which is rather unusual for this system, could reflect a functional adaptation to a role in B. bacteriovorus predation. Finally, I investigated the function of T4aP in B. bacteriovorus through targeted genetic perturbations. Following the impossibility of deleting T4aP components in B. bacteriovorus, I optimized a targeted transcriptional repression approach using an inducible CRISPRi system. This approach demonstrates that loss of T4aP leads to significant predation defects at the population level, characterized by attachment defects at the single-cell scale. These results confirm the hypothesis that T4aP plays an important role in the recognition and/or attachment phase of the predator to its prey, although its precise function remains to be determined. Collectively, this study establishes the importance of T4aP in B. bacteriovorus predation and provides new insights into its function and subcellular dynamics. These findings invite future investigations to determine the mechanisms regulating the localization of the T4aP machinery as well as the precise function of this structure. These results echo other studies describing the use of macromolecular machineries present in the envelope of other bacterial predators that mediate various roles in their predation. These structures thus appear to have been adapted and integrated by numerous predatory bacteria to serve their predatory strategy.