Ryan P. Baker

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.

A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens.

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three‐dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.

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

Significance Vibrio cholerae, the causative agent of the diarrheal disease cholera, uses the syringe-like type VI secretion system (T6SS) to pierce adjacent cells. To investigate the role of the T6SS in invasion of intestines already occupied by symbiotic microbes, we genetically engineered V. cholerae strains and performed live 3D imaging in zebrafish to find that V. cholerae can expel a resident bacterial species in a T6SS-dependent manner. Surprisingly, the T6SS acts primarily to increase the strength of gut contractions, rather than directly killing the bacterial competitor. Deletion of an actin cross-linking domain from the T6SS returned gut activity to normal and eliminated V. cholerae’s competitive advantage. These findings reveal a strategy by which pathogens can manipulate host biomechanics to redefine gut communities. 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.

Image velocimetry and spectral analysis enable quantitative characterization of larval zebrafish gut motility

Normal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Previous image analysis approaches have successfully extracted properties related to the temporal frequency of motility modes, but robust measures of contraction magnitude, especially from in vivo image data, remain challenging to obtain.

Host Gut Motility and Bacterial Competition Drive Instability in 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 we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within the vertebrate gut. We observed that the interplay between each population and the gut environment produced distinct spatiotemporal patterns. Consequently, one species dominates while the other experiences dramatic collapses that are well fit by a stochastic mathematical model. Modeling revealed that 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 mutation in the Hirschsprung disease locus ret. 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 along with bacterial competition drives population dynamics within the gut. In addition, this work highlights the capacity of the enteric nervous system to affect stability of gut microbiota constituents, demonstrating that the ‘gut-brain axis’ is bidirectional. Ultimately, these findings will help inform disease mitigation strategies focused on engineering the intestinal ecosystem.

The Vibrio cholerae Type VI Secretion System Can Modulate Host Intestinal Mechanics to Displace Commensal Gut Bacteria

Host-associated microbiota help defend against bacterial pathogens; the mechanisms that pathogens possess to overcome this defense, however, 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 mono-colonized by commensal 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 Aeromonas, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident commensal by the host. Deleting an actin crosslinking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakening V. cholerae′s ability to kill Aeromonas in vitro. Our finding that bacteria can manipulate host physiology to influence inter-microbial competition has implications for both pathogenesis and microbiome engineering.

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.