Automated high-throughput light-sheet fluorescence microscopy of larval zebrafish.

Light sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.

The Vibrio cholerae type VI secretion system can modulate host intestinal mechanics to displace gut bacterial symbionts.

Host-associated microbiota help defend against bacterial pathogens; however, the mechanisms by which pathogens overcome this defense remain largely unknown. We developed a zebrafish model and used live imaging to directly study how the human pathogen Vibrio cholerae invades the intestine. The gut microbiota of fish monocolonized by symbiotic strain Aeromonas veronii was displaced by V. cholerae expressing its type VI secretion system (T6SS), a syringe-like apparatus that deploys effector proteins into target cells. Surprisingly, displacement was independent of T6SS-mediated killing of A. veronii, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident microbiota by the host. Deleting an actin cross-linking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakening V. cholerae's ability to kill A. veronii in vitro. Our finding that bacteria can manipulate host physiology to influence intermicrobial competition has implications for both pathogenesis and microbiome engineering.

Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota.

The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this "microbial organ" for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host-microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.