Swimming motility of a gut bacterial symbiont promotes resistance to intestinal expulsion and enhances inflammation.

Some of the densest microbial ecosystems in nature thrive within the intestines of humans and other animals. To protect mucosal tissues and maintain immune tolerance, animal hosts actively sequester bacteria within the intestinal lumen. In response, numerous bacterial pathogens and pathobionts have evolved strategies to subvert spatial restrictions, thereby undermining immune homeostasis. However, in many cases, it is unclear how escaping host spatial control benefits gut bacteria and how changes in intestinal biogeography are connected to inflammation. A better understanding of these processes could uncover new targets for treating microbiome-mediated inflammatory diseases. To this end, we investigated the spatial organization and dynamics of bacterial populations within the intestine using larval zebrafish and live imaging. We discovered that a proinflammatory Vibrio symbiont native to zebrafish governs its own spatial organization using swimming motility and chemotaxis. Surprisingly, we found that Vibrio's motile behavior does not enhance its growth rate but rather promotes its persistence by enabling it to counter intestinal flow. In contrast, Vibrio mutants lacking motility traits surrender to host spatial control, becoming aggregated and entrapped within the lumen. Consequently, nonmotile and nonchemotactic mutants are susceptible to intestinal expulsion and experience large fluctuations in absolute abundance. Further, we found that motile Vibrio cells induce expression of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) in gut-associated macrophages and the liver. Using inducible genetic switches, we demonstrate that swimming motility can be manipulated in situ to modulate the spatial organization, persistence, and inflammatory activity of gut bacterial populations. Together, our findings suggest that host spatial control over resident microbiota plays a broader role in regulating the abundance and persistence of gut bacteria than simply protecting mucosal tissues. Moreover, we show that intestinal flow and bacterial motility are potential targets for therapeutically managing bacterial spatial organization and inflammatory activity within the gut.

Disaggregation as an interaction mechanism among intestinal bacteria.

The gut microbiome contains hundreds of interacting species that together influence host health and development. The mechanisms by which intestinal microbes can interact, however, remain poorly mapped and are often modeled as spatially unstructured competitions for chemical resources. Recent imaging studies examining the zebrafish gut have shown that patterns of aggregation are central to bacterial population dynamics. In this study, we focus on bacterial species of genera Aeromonas and Enterobacter. Two zebrafish gut-derived isolates, Aeromonas ZOR0001 (AE) and Enterobacter ZOR0014 (EN), when mono-associated with the host, are highly aggregated and located primarily in the intestinal midgut. An Aeromonas isolate derived from the commensal strain, Aeromonas-MB4 (AE-MB4), differs from the parental strain in that it is composed mostly of planktonic cells localized to the anterior gut. When challenged by AE-MB4, clusters of EN rapidly fragment into non-motile, slow-growing, dispersed individual cells with overall abundance two orders of magnitude lower than the mono-association value. In the presence of a certain set of additional gut bacterial species, these effects on EN are dampened. In particular, if AE-MB4 invades an already established multi-species community, EN persists in the form of large aggregates. These observations reveal an unanticipated competition mechanism based on manipulation of bacterial spatial organization, namely dissolution of aggregates, and provide evidence that multi-species communities may facilitate stable intestinal co-existence.

Sublethal antibiotics collapse gut bacterial populations by enhancing aggregation and expulsion.

Antibiotics induce large and highly variable changes in the intestinal microbiome even at sublethal concentrations, through mechanisms that remain elusive. Using gnotobiotic zebrafish, which allow high-resolution examination of microbial dynamics, we found that sublethal doses of the common antibiotic ciprofloxacin cause severe drops in bacterial abundance. Contrary to conventional views of antimicrobial tolerance, disruption was more pronounced for slow-growing, aggregated bacteria than for fast-growing, planktonic species. Live imaging revealed that antibiotic treatment promoted bacterial aggregation and increased susceptibility to intestinal expulsion. Intestinal mechanics therefore amplify the effects of antibiotics on resident bacteria. Microbial dynamics are captured by a biophysical model that connects antibiotic-induced collapses to gelation phase transitions in soft materials, providing a framework for predicting the impact of antibiotics on the intestinal microbiome.

Assessing the use of ellipsoidal microparticles for determining lipid membrane viscosity.

The viscosity of lipid membranes sets the timescales of membrane-associated motions, whether driven or diffusive, and therefore influences the dynamics of a wide range of cellular processes. Techniques to measure membrane viscosity remain sparse, however, and reported measurements to date, even of similar systems, give viscosity values that span orders of magnitude. To address this, we improve a method based on measuring both the rotational and translational diffusion of membrane-anchored microparticles and apply this approach and one based on tracking the motion of phase-separated lipid domains to the same system of phase-separated giant vesicles. We find good agreement between the two methods, with inferred viscosities within a factor of two of each other. Our single-particle tracking technique uses ellipsoidal microparticles, and we show that the extraction of physically meaningful viscosity values from their motion requires consideration of their anisotropic shape. The validation of our method on phase-separated membranes makes possible its application to other systems, which we demonstrate by measuring the viscosity of bilayers composed of lipids with different chain lengths ranging from 14 to 20 carbon atoms, revealing a very weak dependence of two-dimensional viscosity on lipid size. The experimental and analysis methods described here should be generally applicable to a variety of membrane systems, both reconstituted and cellular.

Experimental bacterial adaptation to the zebrafish gut reveals a primary role for immigration.

All animals live in intimate association with microorganisms that profoundly influence their health and development, yet the traits that allow microorganisms to establish and maintain host associations are not well understood. To date, most investigations aimed at identifying traits required for host association have focused on intrahost niches. Consequently, little is known about the relative contribution of extrahost factors such as environmental growth and survival and immigration into hosts from the external environment, as promoters of host association. To address this, we developed a tractable experimental evolution system that investigates both intra- and extrahost factors contributing to bacterial adaptation to the vertebrate gut. We passaged replicate lines of a zebrafish bacterial isolate, Aeromonas veronii, through populations of germ-free larval zebrafish (Danio rerio), each time using gut-associated Aeromonas populations to inoculate the aquatic environment of the next zebrafish population. We observed rapid increased adaptation to the host in all replicate lines. The initial adaptations present in early-evolved isolates did not increase intrahost fitness but rather enhanced both immigration from the environment and interhost transmission. Only in later-evolved isolates did we find evidence for intrahost-specific adaptations, as demonstrated by comparing their competitive fitness in the host genotype to which they evolved to that in a different genotype. Our results show how selection for bacterial transmission between hosts and their environment can shape bacterial-host association. This work illuminates the nature of selective forces present in host-microbe systems and reveals specific mechanisms of increased host association. Furthermore, our findings demonstrate that the entire host-microbe-environment system must be considered when identifying microbial traits that contribute to host adaptation.

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.

Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.

Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.

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.

Performance of convolutional neural networks for identification of bacteria in 3D microscopy datasets.

Three-dimensional microscopy is increasingly prevalent in biology due to the development of techniques such as multiphoton, spinning disk confocal, and light sheet fluorescence microscopies. These methods enable unprecedented studies of life at the microscale, but bring with them larger and more complex datasets. New image processing techniques are therefore called for to analyze the resulting images in an accurate and efficient manner. Convolutional neural networks are becoming the standard for classification of objects within images due to their accuracy and generalizability compared to traditional techniques. Their application to data derived from 3D imaging, however, is relatively new and has mostly been in areas of magnetic resonance imaging and computer tomography. It remains unclear, for images of discrete cells in variable backgrounds as are commonly encountered in fluorescence microscopy, whether convolutional neural networks provide sufficient performance to warrant their adoption, especially given the challenges of human comprehension of their classification criteria and their requirements of large training datasets. We therefore applied a 3D convolutional neural network to distinguish bacteria and non-bacterial objects in 3D light sheet fluorescence microscopy images of larval zebrafish intestines. We find that the neural network is as accurate as human experts, outperforms random forest and support vector machine classifiers, and generalizes well to a different bacterial species through the use of transfer learning. We also discuss network design considerations, and describe the dependence of accuracy on dataset size and data augmentation. We provide source code, labeled data, and descriptions of our analysis pipeline to facilitate adoption of convolutional neural network analysis for three-dimensional microscopy data.

Bacterial Cohesion Predicts Spatial Distribution in the Larval Zebrafish Intestine.

Are there general biophysical relationships governing the spatial organization of the gut microbiome? Despite growing realization that spatial structure is important for population stability, interbacterial competition, and host functions, it is unclear in any animal gut whether such structure is subject to predictive, unifying rules or if it results from contextual, species-specific behaviors. To explore this, we used light sheet fluorescence microscopy to conduct a high-resolution comparative study of bacterial distribution patterns throughout the entire intestinal volume of live, larval zebrafish. Fluorescently tagged strains of seven bacterial symbionts, representing six different species native to zebrafish, were each separately monoassociated with animals that had been raised initially germ-free. The strains showed large differences in both cohesion-the degree to which they auto-aggregate-and spatial distribution. We uncovered a striking correlation between each strain's mean position and its cohesion, whether quantified as the fraction of cells existing as planktonic individuals, the average aggregate size, or the total number of aggregates. Moreover, these correlations held within species as well; aggregates of different sizes localized as predicted from the pan-species observations. Together, our findings indicate that bacteria within the zebrafish intestine are subject to generic processes that organize populations by their cohesive properties. The likely drivers of this relationship-peristaltic fluid flow, tubular anatomy, and bacterial growth and aggregation kinetics-are common throughout animals. We therefore suggest that the framework introduced here of biophysical links between bacterial cohesion and spatial organization should be useful for directing explorations in other host-microbe systems, formulating detailed models that can quantitatively map onto experimental data, and developing new tools that manipulate cohesion to engineer microbiome function.

Passive and Active Microrheology of the Intestinal Fluid of the Larval Zebrafish.

The fluids of the intestine serve as a physical barrier to pathogens, a medium for the diffusion of nutrients and metabolites, and an environment for commensal microbes. The rheological properties of intestinal mucus have therefore been the subject of many investigations, thus far limited to in vitro studies due to the difficulty of measurement in the natural context of the gut. This limitation especially hinders our understanding of how the gut microbiota interact with the intestinal space, since examination of this calls not only for in vivo measurement techniques, but for techniques that can be applied to model organisms in which the microbial state of the gut can be controlled. We have addressed this challenge with two complementary approaches. We performed passive microrheological measurements using thermally driven nanoparticles and active microrheology using micron-scale ellipsoidal magnetic microparticles, in both cases using light-sheet fluorescence microscopy to optically access the intestinal bulb of the larval zebrafish, a model vertebrate. We present viscosity measurements in germ-free animals (devoid of gut microbes), animals colonized by a single bacterial species, and conventionally reared animals, and find that in all cases, the mucin-rich intestinal liquid is well described as a Newtonian fluid. Surprisingly, despite known differences in the number of secretory cells in germ-free zebrafish and their conventional counterparts, the fluid viscosity for these two groups is very similar, as measured with either technique. Our study provides, to our knowledge, the first in vivo microrheological measurements of the intestinal space in living animals, and we comment on its implications for timescales of host-microbe interactions in the gut.

Tension Independence of Lipid Diffusion and Membrane Viscosity.

The diffusion of biomolecules at lipid membranes is governed by the viscosity of the underlying two-dimensionally fluid lipid bilayer. For common three-dimensional fluids, viscosity can be modulated by hydrostatic pressure, and pressure-viscosity data have been measured for decades. Remarkably, the two-dimensional analogue of this relationship, the dependence of molecular mobility on tension, has to the best of our knowledge never been measured for lipid bilayers, limiting our understanding of cellular mechanotransduction as well as the fundamental fluid mechanics of membranes. Here we report both molecular-scale and mesoscopic measures of fluidity in giant lipid vesicles as a function of mechanical tension applied using micropipette aspiration. Both molecular-scale data, from fluorescence recovery after photobleaching, and micron-scale data, from tracking the diffusion of phase-separated domains, show a surprisingly weak dependence of viscosity on tension, in contrast to predictions of recent molecular dynamics simulations, highlighting fundamental gaps in our understanding of membrane fluidity.

Two-Point Microrheology of Phase-Separated Domains in Lipid Bilayers.

Though the importance of membrane fluidity for cellular function has been well established for decades, methods for measuring lipid bilayer viscosity remain challenging to devise and implement. Recently, approaches based on characterizing the Brownian dynamics of individual tracers such as colloidal particles or lipid domains have provided insights into bilayer viscosity. For fluids in general, however, methods based on single-particle trajectories provide a limited view of hydrodynamic response. The technique of two-point microrheology, in which correlations between the Brownian dynamics of pairs of tracers report on the properties of the intervening medium, characterizes viscosity at length-scales that are larger than that of individual tracers and has less sensitivity to tracer-induced distortions, but has never been applied to lipid membranes. We present, to our knowledge, the first two-point microrheological study of lipid bilayers, examining the correlated motion of domains in phase-separated lipid vesicles and comparing one- and two-point results. We measure two-point correlation functions in excellent agreement with the forms predicted by two-dimensional hydrodynamic models, analysis of which reveals a viscosity intermediate between those of the two lipid phases, indicative of global fluid properties rather than the viscosity of the local neighborhood of the tracer.

A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

Rapid, accurate particle tracking by calculation of radial symmetry centers.

I introduce an algorithm for subpixel localization of imaged objects based on an analytic, non-iterative calculation of the best-fit radial symmetry center. This approach yields tracking accuracies that are near theoretical limits, similarly to Gaussian fitting, but with orders-of-magnitude faster execution time, lower sensitivity to nearby particles and applicability to any radially symmetric intensity distribution. I demonstrate the method with several types of data, including super-resolution microscopy images.

Superresolution localization methods.

Superresolution localization microscopy methods produce nanoscale images via a combination of intermittently active fluorescent probes and algorithms that can precisely determine the positions of these probes from single-molecule or few-molecule images. These algorithms vary widely in their underlying principles, complexity, and accuracy. In this review, we begin by surveying the principles of localization microscopy and describing the fundamental limits to localization precision. We then examine several different families of fluorophore localization algorithms, comparing their complexity, performance, and range of applicability (e.g., whether they require particular types of experimental information, are optimized for specific situations, or are more general). Whereas our focus is on the localization of single isotropic emitters in two dimensions, we also consider oriented dipoles, three-dimensional localization, and algorithms that can handle overlapping images of several nearby fluorophores. Throughout the review, we try to highlight practical advice for users of fluorophore localization algorithms, as well as open questions.

Measuring lipid membrane viscosity using rotational and translational probe diffusion.

The two-dimensional fluidity of lipid bilayers enables the motion of membrane-bound macromolecules and is therefore crucial to biological function. Microrheological methods that measure fluid viscosity via the translational diffusion of tracer particles are challenging to apply and interpret for membranes, due to uncertainty about the local environment of the tracers. Here, we demonstrate a new technique in which determination of both the rotational and translational diffusion coefficients of membrane-linked particles enables quantification of viscosity, measurement of the effective radii of the tracers, and assessment of theoretical models of membrane hydrodynamics. Surprisingly, we find a wide distribution of effective tracer radii, presumably due to a variable number of lipids linked to each tracer particle. Furthermore, we show for the first time that a protein involved in generating membrane curvature, the vesicle trafficking protein Sar1p, dramatically increases membrane viscosity. Using the rheological method presented here, therefore, we are able to reveal a class of previously unknown couplings between protein activity and membrane mechanics.

Robust measurement of membrane bending moduli using light sheet fluorescence imaging of vesicle fluctuations.

The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons between disparate techniques applied to the same systems. We introduce here the use of selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) to image thermal fluctuations of giant vesicles. The optical sectioning of SPIM enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For both lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, which lowers membrane rigidity in a concentration-dependent manner, we show that the resulting bending modulus values are in close agreement with those derived from an independent assay based on membrane tether pulling. We also show that the fluctuation spectra of vesicles bound by the mammalian Sar1A protein, which stiffens membranes at high concentrations, are not well fit by a model of homogeneous quasi-spherical vesicles, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties induced by protein assemblies.

Phospholipid bilayers are viscoelastic.

Lipid bilayers provide the structural framework for cellular membranes, and their character as two-dimensional fluids enables the mobility of membrane macromolecules. Though the existence of membrane fluidity is well established, the nature of this fluidity remains poorly characterized. Three-dimensional fluids as diverse as chocolates and cytoskeletal networks show a rich variety of Newtonian and non-Newtonian dynamics that have been illuminated by contemporary rheological techniques. Applying particle-tracking microrheology to freestanding phospholipid bilayers, we find that the membranes are not simply viscous but rather exhibit viscoelasticity, with an elastic modulus that dominates the response above a characteristic frequency that diverges at the fluid-gel (L(α) - L(ß)) phase-transition temperature. These findings fundamentally alter our picture of the nature of lipid bilayers and the mechanics of membrane environments.

A mucin-regulated adhesin determines the spatial organization and inflammatory character of a bacterial symbiont in the vertebrate gut.

In a healthy gut, microbes are often aggregated with host mucus, yet the molecular basis for this organization and its impact on intestinal health are unclear. Mucus is a viscous physical barrier separating resident microbes from epithelia, but it also provides glycan cues that regulate microbial behaviors. Here, we describe a mucin-sensing pathway in an Aeromonas symbiont of zebrafish, Aer01. In response to the mucin-associated glycan N-acetylglucosamine, a sensor kinase regulates the expression of an aggregation-promoting adhesin we named MbpA. Upon MbpA disruption, Aer01 colonizes to normal levels but is largely planktonic and more pro-inflammatory. Increasing cell surface MbpA rescues these traits. MbpA-like adhesins are common in human-associated bacteria, and the expression of an Akkermansia muciniphila MbpA-like adhesin in MbpA-deficient Aer01 restores lumenal aggregation and reverses its pro-inflammatory character. Our work demonstrates how resident bacteria use mucin glycans to modulate behaviors congruent with host health.