Integrating biological knowledge for mechanistic inference in the host-associated microbiome.

Advances in high-throughput technologies have enhanced our ability to describe microbial communities as they relate to human health and disease. Alongside the growth in sequencing data has come an influx of resources that synthesize knowledge surrounding microbial traits, functions, and metabolic potential with knowledge of how they may impact host pathways to influence disease phenotypes. These knowledge bases can enable the development of mechanistic explanations that may underlie correlations detected between microbial communities and disease. In this review, we survey existing resources and methodologies for the computational integration of broad classes of microbial and host knowledge. We evaluate these knowledge bases in their access methods, content, and source characteristics. We discuss challenges of the creation and utilization of knowledge bases including inconsistency of nomenclature assignment of taxa and metabolites across sources, whether the biological entities represented are rooted in ontologies or taxonomies, and how the structure and accessibility limit the diversity of applications and user types. We make this information available in a code and data repository at: https://github.com/lozuponelab/knowledge-source-mappings. Addressing these challenges will allow for the development of more effective tools for drawing from abundant knowledge to find new insights into microbial mechanisms in disease by fostering a systematic and unbiased exploration of existing information.

Complete Genome Sequence of a Staphylococcus epidermidis Isolate from Healthy Skin with Upregulation of Protease EcpA.

Staphylococcus epidermidis is a ubiquitous skin commensal that has the potential to become pathogenic and cause disease. Here, we report the complete genome sequence of a S. epidermidis strain isolated from adult healthy skin that shows high expression of the virulence factor extracellular cysteine protease A (EcpA).

BefA, a microbiota-secreted membrane disrupter, disseminates to the pancreas and increases ß cell mass.

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.

Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health.

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.

Pseudocapillaria tomentosa, Mycoplasma spp., and Intestinal Lesions in Experimentally Infected Zebrafish Danio rerio.

Intestinal neoplasms and preneoplastic lesions are common in zebrafish research facilities. Previous studies have demonstrated that these neoplasms are caused by a transmissible agent, and two candidate agents have been implicated: a Mycoplasma sp. related to Mycoplasma penetrans and the intestinal parasitic nematode, Pseudocapillaria tomentosa, and both agents are common in zebrafish facilities. To elucidate the role of these two agents in the occurrence and severity of neoplasia and other intestinal lesions, we conducted two experimental inoculation studies. Exposed fish were examined at various time points over an 8-month period for intestinal histopathologic changes and the burden of Mycoplasma and nematodes. Fish exposed to Mycoplasma sp. isolated from zebrafish were associated with preneoplastic lesions. Fish exposed to the nematode alone or with the Mycoplasma isolate developed severe lesions and neoplasms. Both inflammation and neoplasm scores were associated with an increase in Mycoplasma burden. These results support the conclusions that P. tomentosa is a strong promoter of intestinal neoplasms in zebrafish and that Mycoplasma alone can also cause intestinal lesions and accelerate cancer development in the context of nematode infection.

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.

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.

Bacteria evoke alarm behaviour in zebrafish.

When injured, fish release an alarm substance (Schreckstoff) that elicits fear in members of their shoal. Although Schreckstoff has been proposed to be produced by club cells in the skin, several observations indicate that these giant cells function primarily in immunity. Previous data indicate that the alarm substance can be isolated from mucus. Here we show that mucus, as well as bacteria, are transported from the external surface into club cells, by cytoplasmic transfer or invasion of cells, including neutrophils. The presence of bacteria inside club cells raises the possibility that the alarm substance may contain a bacterial component. Indeed, lysate from a zebrafish Staphylococcus isolate is sufficient to elicit alarm behaviour, acting in concert with a substance from fish. These results suggest that Schreckstoff, which allows one individual to unwittingly change the emotional state of the surrounding population, derives from two kingdoms and is associated with processes that protect the host from bacteria.

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.

Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages.

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.