Bacteria can form multi-cellular communities in which individual cells are protected from environmental insults such as antibiotics by virtue of being [1] encased in a protective matrix comprised of polysaccharides and other macromolecules and [2] physiologically distinct from free-living, planktonic cells. Biofilm formation enhances the ability of bacteria to colonize surfaces, including host tissues and abiotic surfaces such as medical implants, and seeds subsequent infections at distal locations through dispersal processes. As a result of these characteristics, bacteria in biofilms are responsible for the majority of hospital-acquired infections and thus understanding how biofilms form and disperse from such biofilms is critical. Although numerous animal models of biofilm formation have been developed, few, if any, permit visual examination of biofilm formation and dispersal events as well as a quantitative analysis of subsequent colonization outcomes. One such robust model, however, can be found in the Vibrio fischeri-squid (Euprymna scolopes) symbiosis. To colonize, V. fischeri first forms a biofilm on the surface of the symbiotic organ, then disperses from it to enter and ultimately colonize sites deep within this organ. Our work has shown that genes required for biofilm formation in laboratory culture are similarly required for host-associated (HA) biofilms and colonization, while genetic changes that enhance biofilm formation in the lab also strikingly enhance HA biofilms and colonization. This strong correlation affords us an exceptional opportunity to develop and test hypotheses about the mechanisms of HA-biofilms, dispersal and subsequent colonization. Our work has revealed that HA biofilms and colonization depend on syp, an 18-gene locus involved in production of SYP polysaccharide, and on multiple sensor kinases and response regulators that control syp transcription and post-transcriptional events. We have recently identified calcium (Ca2+) and nitric oxide (NO) as a strong inducer and inhibitor, respectively, of biofilm formation. Ca2+ is a physiologically relevant signal that appears to affect numerous processes, but how it does so is as-yet unknown. NO, which is produced by the squid and known to influence HA-biofilms, likely impacts one of the sensor kinases required for syp transcription, and we propose to evaluate the underlying mechanisms. We are also poised to identify other physiological signals that promote/inhibit HA biofilms. We have recently uncovered conditions in which dispersal can be visualized in laboratory culture, and have observed that V. fischeri undergoes multiple rounds of formation and dispersal in fully-grown cultures, a result that suggests control by post-transcriptional mechanisms. We have begun to investigate that process by identifying a set of genes involved in controlling dispersal. We propose to develop a mechanistic understanding of these dispersal genes and the factors that control dispersal events as well as to search for others that we predict to exist. We anticipate that this work will provide insights into the mechanisms by which bacteria respond to their environment and transition in and out of multi-cellular communities within an animal host.
Many if not most bacterial infectious diseases as well as the normal interactions between a human or animal host and its microbiome occur in the context of bacterial biofilms, communities of bacteria protected from host defenses as well as therapeutic antimicrobials. We study the symbiosis between the bacterium Vibrio fischeri and a squid as a simple animal model that permits us to readily visualize host (animal)-associated biofilm formation and dispersal, an event that permits bacteria to spread, and evaluate the underlying mechanisms. We are poised here to investigate signals and processes that promote host-associated biofilms and dispersal, and anticipate that our ability to readily investigate how bacteria enter and leave biofilms in the context of an animal will promote our ability to treat infectious biofilms in the context of human disease.
