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Agrobacterium tumefaciens is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between A. tumefaciens and host cells promotes infection efficacy. Thus, improving the strength and specificity of A. tumefaciens to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here we demonstrate a methodology for engineering genetically-encoded exogeneous adhesins at the surface of A. tumefaciens. We identified an autotransporter gene we named Aat, that is predicted to show canonical β-barrel and passenger domains. We engineered the β-barrel scaffold and linker (Aatβ) to display synthetic adhesins susceptible to rewire A. tumefaciens to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire A. tumefaciens adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic A. tumefaciens adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering A. tumefaciens adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.

Bacterial gut commensals experience a biologically and physically complex mucosal environment. While many chemical factors mediate the composition and structure of these microbial communities, less is known about the role of mechanics. Here, we demonstrate that fluid flow impacts the spatial organization and composition of gut biofilm communities by shaping how different species interact metabolically. We first demonstrate that a model community composed of Bacteroides thetaiotaomicron (Bt) and Bacteroides fragilis (Bf), two representative human commensals, can form robust biofilms in flow. We identified dextran as a polysaccharide readily metabolized by Bt but not Bf, but whose fermentation generates a public good enabling Bf growth. By combining simulations with experiments, we demonstrate that in flow, Bt biofilms share dextran metabolic by-products, promoting Bf biofilm formation. By transporting this public good, flow structures the spatial organization of the community, positioning the Bf population downstream from Bt. We show that sufficiently strong flows abolish Bf biofilm formation by limiting the effective public good concentration at the surface. Physical factors such as flow may therefore contribute to the composition of intestinal microbial communities, potentially impacting host health., Proceedings of the National Academy of Sciences of the United States of America, 120 (25), ISSN:10916490, ISSN:0027-8424, ISSN:1091-6490

Pathogen attachment to host tissue is critical in the progress of many infections. Bacteria use adhesionin vivoto promote colonization and regulate the deployment of contact-dependent virulence traits. To specifically target host cells, they decorate themselves with adhesins, proteins that bind to mammalian cell surface receptors. One common assumption is that adhesin-receptor interactions entirely govern bacterial attachment. However, how adhesins engage with their receptors in anin vivo-like context remains unclear, in particular under the influence of a heterogeneous mechanical microenvironment. We here investigate the biophysical processes governing bacterial adhesion to host cells using a tunable adhesin-receptor system. By dynamically visualizing attachment, we found that bacterial adhesion to host cell surface, unlike adhesion to inert surfaces, involves two consecutive steps. Bacteria initially attach to their host without engaging adhesins. This step lasts about one minute during which bacteria can easily detach. We found that at this stage, the glycocalyx, a layer of glycosylated proteins and lipids, shields the host cell by keeping adhesin away from their receptor ligand. In a second step, adhesins engage with their target receptors to strengthen attachment for minutes to hours. The active properties of the membrane, endowed by the actin cytoskeleton, strengthen specific adhesion. Altogether, our results demonstrate that adhesin-ligand binding is not the sole regulator of bacterial adhesion. In fact, the host cell’s mechanical microenvironment relatively strongly mediated host-bacteria physical interactions, thereby playing an essential role in the onset of infection.