Identification and characterization of a skin microbiome on Caenorhabditis elegans suggests environmental microbes confer cuticle protection.

In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple "skin microbiome." We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101, are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal's gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts. IMPORTANCE: The genetic model organism C. elegans has recently emerged as a tool for understanding host-microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans, and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

Intestine-to-neuronal signaling alters risk-taking behaviors in food-deprived Caenorhabditis elegans.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Microbial Mind Control.

Every animal is in constant communication with populations of microbes. In a recent study, O'Donnell and colleagues (2020) uncover an inter-domain conversation, defining a relationship in which a non-pathogenic microbe directly synthesizes a signal that alters host behavior for a mutually beneficial outcome.

Epithelial delamination is protective during pharmaceutical-induced enteropathy.

Intestinal epithelial cell (IEC) shedding is a fundamental response to intestinal damage, yet underlying mechanisms and functions have been difficult to define. Here we model chronic intestinal damage in zebrafish larvae using the nonsteroidal antiinflammatory drug (NSAID) Glafenine. Glafenine induced the unfolded protein response (UPR) and inflammatory pathways in IECs, leading to delamination. Glafenine-induced inflammation was augmented by microbial colonization and associated with changes in intestinal and environmental microbiotas. IEC shedding was a UPR-dependent protective response to Glafenine that restricts inflammation and promotes animal survival. Other NSAIDs did not induce IEC delamination; however, Glafenine also displays off-target inhibition of multidrug resistance (MDR) efflux pumps. We found a subset of MDR inhibitors also induced IEC delamination, implicating MDR efflux pumps as cellular targets underlying Glafenine-induced enteropathy. These results implicate IEC delamination as a protective UPR-mediated response to chemical injury, and uncover an essential role for MDR efflux pumps in intestinal homeostasis.

Intestinal Serum amyloid A suppresses systemic neutrophil activation and bactericidal activity in response to microbiota colonization.

The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions remain poorly defined. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory markers and bactericidal activity. Saa's effects on neutrophils depended on microbiota colonization, suggesting this protein mediates the microbiota's effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.

A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.

Live Imaging of Host-Pathogen Interactions in Zebrafish Larvae.

Zebrafish larvae are a powerful platform for studying the innate immune response to infection. The small size and optical transparency of larval zebrafish allow for multiple subject, multidimensional, and longitudinal imaging experiments. This chapter describes protocols for infecting zebrafish larvae with their natural pathogen Mycobacterium marinum, rapid short-term imaging, long-term extended imaging, and drug treatment assays. These protocols can be easily adapted to image and manipulate host interactions with other pathogens.

Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

Adventures within the speckled band: heterogeneity, angiogenesis, and balanced inflammation in the tuberculous granuloma.

Recent work in a variety of animal models, including mice, zebrafish, and macaques, as well as in humans, has led to a reassessment of several tenets of mycobacterial infection. In this review, we describe new findings about the composition and dynamics of the tuberculous granuloma, the central host structure in mycobacterial infection, as well as inflammatory mediators that drive a successful anti-microbial response on one hand and pathological inflammation on the other. We highlight granuloma heterogeneity that emerges in the context of infection, the functional consequences of angiogenesis in tuberculous granulomas, and data that balanced inflammation in humans, with a central role for tumor necrosis factor, appears to play a key role in optimal defense against mycobacterial infection. These findings have suggested new and specific host-directed therapies that await further clinical exploration.