A Role for Secreted Immune Effectors in Microbial Biofilm Formation Revealed by Simple In Vitro Assays

The formation of biofilms is critical for the successful and stable colonization of mucosal surfaces by microbes, which often build three-dimensional environments by exuding exopolysaccharides and other macromolecules such as proteins, lipids, and even DNA. It is not just bacteria, but fungi such as yeast, that form these adherent interacting communities. Historically, biofilms have been studied in the context of pathogenesis, but only recently it has been recognized that important relationships among members of host-associated microbiomes are maintained within the context of biofilms. Host immune responses impact biofilm formation in various ways; for example, it is likely that formation of stable biofilms by non-pathogens improves barrier defenses by not just filling available niche spaces but also by helping to ward off pathogens directly. Recently, it was found that soluble immune effector molecules such as immunoglobulin A (IgA) in mammals serve essential roles in modulating complex biofilm communities in ways that benefit the host. Additional lines of evidence from other secreted immune effectors, such as the variable region-containing chitin-binding proteins (VCBPs) in protochordates, now suggest that this phenomenon is much more widespread than previously recognized. The activity of these immune molecules also likely serves roles beyond those of simple defense strategies; rather, they may be improving the outcome of symbiotic interactions benefiting the host. Thus, traditional immune assays that are aimed at studying the function of secreted immune effectors, such as agglutination assays, should take into account the possibility that the first observation may not be the last if the microbes under study are not directly killed. Here, we describe a series of simple approaches to characterize biofilm formation when bacteria (or yeast) are cultured in the presence of a secreted immune effector. To model this approach, we use microbes isolated from the gut of Ciona robusta, each grown in the presence or absence of VCBPs. The approaches defined here are amenable to diverse model systems and their microbes.