BELOIN, C. | Search bioRxiv

bioRxiv2020

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

microbiology↗

bioRxiv2020

Capsular polysaccharides cross-regulation modulates Bacteroides thetaiotaomicron biofilm formation

Bacteroides thetaiotaomicron is one of the most abundant gut symbiont species, whose contribution to host health through its ability to degrade diet polysaccharides and mature the immune system is under untense scrutiny. By contrast, adhesion and biofilm formation, which are potentially involved in gut colonization, microbiota structure and stability, have hardly been investigated in this intestinal bacterium. To uncover B. thetaiotaomicron biofilm-related functions, we performed a transposon mutagenesis in the poor biofilm-forming reference strain VPI 5482 and showed that capsule 4, one of the eight B. thetaiotaomicron capsules, hinders biofilm formation. We then showed that the production of capsules 1, 2, 3, 5 and 6 also inhibits biofilm formation and that decreased capsulation of the population correlated with increased biofilm formation, suggesting that capsules could be masking adhesive surface structures. We also showed that, by contrast, capsule 8 displayed intrinsic adhesive properties. Finally, we demonstrated that BT2934, the wzx homolog of B. thetaiotaomicron glycosylation locus, competes with capsule production and therefore impacts its adhesion capacity. This study identified regulation of capsular polysaccharides as a major determinant of B. thetaiotaomicron biofilm formation, providing new insights into how modulation of different B. thetaiotaomicron surface structures affect in vitro biofilm formation.

microbiology↗