RESUMEN
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.
Asunto(s)
Mucinas , Pez Cebra , Animales , Humanos , Mucinas/metabolismo , Bacterias/metabolismo , Polisacáridos/metabolismo , Epitelio/metabolismoRESUMEN
Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. We report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing ß cells during development in gnotobiotic zebrafish and mice. BefA disseminates systemically by multiple anatomic routes to act directly on pancreatic islets. We detail BefA's atomic structure, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand ß cells, whereas the pore-forming host defense protein, Reg3, stimulates ß cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for ß cell expansion during pancreas development, potentially connecting microbiome composition with diabetes risk.
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Diabetes Mellitus , Microbiota , Ratones , Animales , Pez Cebra , Páncreas/metabolismo , Insulina/metabolismo , Diabetes Mellitus/metabolismo , Proteínas/metabolismoRESUMEN
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.
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Microbioma Gastrointestinal , Pez Cebra , Animales , Bacterias , Pez Cebra/microbiologíaRESUMEN
Bacterial motility is a widespread characteristic that can provide several advantages for the cell, allowing it to move towards more favorable conditions and enabling host-associated processes such as colonization. There are different bacterial motility types, and their expression is highly regulated by the environmental conditions. Because of this, methods for studying motility under realistic experimental conditions are required. A wide variety of approaches have been developed to study bacterial motility. Here, we present the most common techniques and recent advances and discuss their strengths as well as their limitations. We classify them as macroscopic or microscopic and highlight the advantages of three-dimensional imaging in microscopic approaches. Lastly, we discuss methods suited for studying motility in bacterial-host interactions, including the use of the zebrafish model.
RESUMEN
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.
Asunto(s)
Membrana Dobles de Lípidos , Difusión , Membranas , ViscosidadRESUMEN
The spatial organization of gut microbiota influences both microbial abundances and host-microbe interactions, but the underlying rules relating bacterial dynamics to large-scale structure remain unclear. To this end, we studied experimentally and theoretically the formation of three-dimensional bacterial clusters, a key parameter controlling susceptibility to intestinal transport and access to the epithelium. Inspired by models of structure formation in soft materials, we sought to understand how the distribution of gut bacterial cluster sizes emerges from bacterial-scale kinetics. Analyzing imaging-derived data on cluster sizes for eight different bacterial strains in the larval zebrafish gut, we find a common family of size distributions that decay approximately as power laws with exponents close to -2, becoming shallower for large clusters in a strain-dependent manner. We show that this type of distribution arises naturally from a Yule-Simons-type process in which bacteria grow within clusters and can escape from them, coupled to an aggregation process that tends to condense the system toward a single massive cluster, reminiscent of gel formation. Together, these results point to the existence of general, biophysical principles governing the spatial organization of the gut microbiome that may be useful for inferring fast-timescale dynamics that are experimentally inaccessible.
The human gut is home to vast numbers of bacteria that grow, compete and cooperate in a dynamic, densely packed space. The spatial arrangement of organisms for example, if they are clumped together or broadly dispersed plays a major role in all ecosystems; but how bacteria are organized in the human gut remains mysterious and difficult to investigate. Zebrafish larvae provide a powerful tool for studying microbes in the gut, as they are optically transparent and anatomically similar to other vertebrates, including humans. Furthermore, zebrafish can be easily manipulated so that one species of bacteria can be studied at a time. To investigate whether individual bacterial species are arranged in similar ways, Scholmann and Parthasarathy exposed zebrafish with no gut bacteria to one of eight different strains. Each species was then monitored using three-dimensional microscopy to see how the population shaped itself into clusters (or colonies). Schlomann and Parthasarathy used this data to build a mathematical model that can predict the size of the clusters formed by different gut bacteria. This revealed that the spatial arrangement of each species depended on the same biological processes: bacterial growth, aggregation and fragmentation of clusters, and expulsion from the gut. These new details about how bacteria are organized in zebrafish may help scientists learn more about gut health in humans. Although it is not possible to peer into the human gut and watch how bacteria behave, scientists could use the same analysis method to study the size of bacterial colonies in fecal samples. This may provide further clues about how microbes are spatially arranged in the human gut and the biological processes underlying this formation.
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Bacterias/crecimiento & desarrollo , Fenómenos Fisiológicos Bacterianos , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Animales , Tracto Gastrointestinal/embriología , Geles , Cinética , Modelos Teóricos , Densidad de Población , Pez Cebra/embriología , Pez Cebra/microbiologíaRESUMEN
The microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health, and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multispecies consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germfree, to allow the introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, interbacterial interactions become weaker, suggesting that higher-order interactions in the vertebrate gut stabilize complex communities.IMPORTANCE Understanding the rules governing the composition of the diverse microbial communities that reside in the vertebrate gut environment will enhance our ability to manipulate such communities for therapeutic ends. Synthetic microbial communities, assembled from specific combinations of microbial species in germfree animals, allow investigation of the fundamental question of whether multispecies community composition can be predicted solely based on the combined effects of interactions between pairs of species. If so, such predictability would enable the construction of communities with desired species from the bottom up. If not, the apparent higher-order interactions imply that emergent community-level characteristics are crucial. Our findings using up to five coexisting native bacterial species in larval zebrafish, a model vertebrate, provide experimental evidence for higher-order interactions and, moreover, show that these interactions promote the coexistence of microbial species in the gut.
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Bacterias/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Simbiosis , Pez Cebra/microbiología , Animales , Bacterias/clasificación , Fenómenos Fisiológicos Bacterianos , Larva/microbiologíaRESUMEN
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.
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Microbioma Gastrointestinal/fisiología , Motilidad Gastrointestinal/fisiología , Intestinos/patología , Locomoción/fisiología , Animales , Animales Modificados Genéticamente , Quimiotaxis/genética , Quimiotaxis/fisiología , Inflamación , Intestinos/microbiología , Locomoción/genética , Macrófagos/metabolismo , Interacciones Microbianas , Mutación , Factor de Necrosis Tumoral alfa/metabolismo , Vibrio/genética , Vibrio/fisiología , Pez Cebra/microbiología , Pez Cebra/fisiologíaRESUMEN
Vast communities of microorganisms inhabit the gastrointestinal tracts of humans and other animals. Understanding their initial development, fluctuations in composition, stability over long times, and responses to transient perturbations - in other words their dynamics - is important both for gaining basic insights into these ecosystems and for rationally manipulating them for therapeutic ends. Gut microbiome dynamics, however, remain poorly understood. We review here studies of gut microbiome dynamics in the presence and absence of external perturbations, noting especially the long timescales associated with overall stability and the short timescales associated with various underlying biological processes. Integrating these disparate timescales, we suggest, is an important goal for future work and is necessary for developing a predictive understanding of microbiome dynamics.
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Microbioma Gastrointestinal , Animales , Humanos , Ratones , Modelos Teóricos , Factores de TiempoRESUMEN
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.
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Antibacterianos/toxicidad , Bacterias/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Animales , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Intestinos/efectos de los fármacos , Intestinos/microbiología , Pez Cebra/microbiologíaRESUMEN
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.
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Bacterias/clasificación , Bacterias/ultraestructura , Imagenología Tridimensional/métodos , Redes Neurales de la Computación , Algoritmos , Animales , Biología Computacional , Bases de Datos Factuales/estadística & datos numéricos , Humanos , Imagenología Tridimensional/estadística & datos numéricos , Intestinos/microbiología , Microscopía Fluorescente , Pseudomonas/ultraestructura , Máquina de Vectores de Soporte , Vibrio/ultraestructura , Pez Cebra/microbiologíaRESUMEN
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.
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Adaptación Biológica/fisiología , Tracto Gastrointestinal/microbiología , Interacciones Microbiota-Huesped/fisiología , Adaptación Biológica/genética , Aeromonas veronii/metabolismo , Aeromonas veronii/fisiología , Animales , Bacterias , Evolución Biológica , Microbioma Gastrointestinal/fisiología , Interacciones Microbiota-Huesped/inmunología , Larva/microbiología , Filogenia , Selección Genética/genética , Selección Genética/fisiología , Pez Cebra/microbiologíaRESUMEN
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.
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Bacterias/patogenicidad , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Intestinos/microbiología , Larva/microbiología , Pez Cebra/microbiología , Animales , Adhesión Bacteriana , Tracto Gastrointestinal/fisiología , Intestinos/fisiología , Análisis Espacio-Temporal , Especificidad de la Especie , Pez Cebra/clasificaciónRESUMEN
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.
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Embrión no Mamífero , Larva , Microscopía Fluorescente/métodos , Pez Cebra , Animales , Imagenología Tridimensional/métodosRESUMEN
Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.
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Técnicas Genéticas , Genoma Bacteriano , Microbiota , Proteobacteria/clasificación , Animales , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Intestinos/microbiología , Fenotipo , Plásmidos , Análisis de Secuencia de ADN , Simbiosis , Pez Cebra/microbiologíaRESUMEN
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.
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Antibiosis/fisiología , Microbioma Gastrointestinal , Sistemas de Secreción Tipo VI/fisiología , Vibrio cholerae/fisiología , Pez Cebra/microbiología , Actinas/fisiología , Aeromonas veronii , Animales , Proteínas Bacterianas/fisiología , Motilidad Gastrointestinal , Vida Libre de Gérmenes , Interacciones Huésped-Patógeno , Simbiosis , Vibrio cholerae/patogenicidadRESUMEN
Microbes often live in dense, dynamic, multi-species communities whose architecture and function are intimately intertwined. Imaging these complex, three-dimensional ensembles presents considerable technical challenges, however. In this review, I describe light sheet fluorescence microscopy, a technique that enables rapid acquisition of three-dimensional images over large fields of view and over long durations, and I highlight recent applications of this method to microbial systems that include artificial closed ecosystems, bacterial biofilms, and gut microbiota. I comment also on the history of light sheet imaging and the many variants of the method. Light sheet techniques have tremendous potential for illuminating the workings of microbial communities, a potential that is just beginning to be realized.
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Imagenología Tridimensional/instrumentación , Luz , Microbiota , Microscopía Fluorescente/métodos , Biopelículas , Microbioma Gastrointestinal , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentaciónRESUMEN
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.
RESUMEN
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.
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Líquido Extracelular/metabolismo , Larva/citología , Reología , Pez Cebra , AnimalesRESUMEN
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.