The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

Complement-ary protection for all ages.

Complement proteins form a proteolytic cascade to clear invading microbes. In a recent issue of Cell, Wu et al. and Xu et al. demonstrate two distinct complement systems operating in the gut, independent of circulating complement, and protecting against intestinal pathogens.

Microbial underdogs: exploring the significance of low-abundance commensals in host-microbe interactions.

Our understanding of host-microbe interactions has broadened through numerous studies over the past decades. However, most investigations primarily focus on the dominant members within ecosystems while neglecting low-abundance microorganisms. Moreover, laboratory animals usually do not have microorganisms beyond bacteria. The phenotypes observed in laboratory animals, including the immune system, have displayed notable discrepancies when compared to real-world observations due to the diverse microbial community in natural environments. Interestingly, recent studies have unveiled the beneficial roles played by low-abundance microorganisms. Despite their rarity, these keystone taxa play a pivotal role in shaping the microbial composition and fulfilling specific functions in the host. Consequently, understanding low-abundance microorganisms has become imperative to unravel true commensalism. In this review, we provide a comprehensive overview of important findings on how low-abundance commensal microorganisms, including low-abundance bacteria, fungi, archaea, and protozoa, interact with the host and contribute to host phenotypes, with emphasis on the immune system. Indeed, low-abundance microorganisms play vital roles in the development of the host's immune system, influence disease status, and play a key role in shaping microbial communities in specific niches. Understanding the roles of low-abundance microbes is important and will lead to a better understanding of the true host-microbe relationships.

Mucin-binding adhesins: A key to unlocking the door of mutualism.

From corals to mammals, mucus is a conserved feature that prevents microbes from accessing the surfaces that produce it. However, interactions between mucus and microbes remain poorly understood. In this issue of Cell Host & Microbe, Smith et al. unveil that mucus binding by bacteria is crucial for host-microbe symbiosis.

Out of destruction comes new growth: Pore-forming antimicrobials make pancreas grow.

Gut-residing bacteria are known to regulate the physiologies of distal organs. However, the mechanism behind the long-distance communication between gut microbes and distal organs remains unknown. In this issue of Cell Metabolism, two studies show that ß cell expansion in the pancreas depends on bacterially induced antimicrobials produced in the gut.

Gut commensals expand vitamin A metabolic capacity of the mammalian host.

Conversion of dietary vitamin A (VA) into retinoic acid (RA) is essential for many biological processes and thus far studied largely in mammalian cells. Using targeted metabolomics, we found that commensal bacteria in the mouse gut lumen produced a high concentration of the active retinoids, all-trans-retinoic acid (atRA) and 13-cis-retinoic acid (13cisRA), as well as the principal circulating retinoid, retinol. Ablation of anerobic bacteria significantly reduced retinol, atRA, and 13cisRA, whereas introducing these bacteria into germ-free mice significantly enhanced retinoids. Remarkably, cecal bacterial supplemented with VA produced active retinoids in vitro, establishing that gut bacteria encode metabolic machinery necessary for multistep conversion of dietary VA into its active forms. Finally, gut bacteria Lactobacillus intestinalis metabolized VA and specifically restored RA levels in the gut of vancomycin-treated mice. Our work establishes vitamin A metabolism as an emergent property of the gut microbiome and lays the groundwork for developing probiotic-based retinoid therapy.

Low abundance members of the gut microbiome exhibit high immunogenicity.

Studies identifying bacterial members that dictate host phenotype have focused mainly on the dominant members, and the role of low abundance microbes in determining host phenotypes and pathogenesis of diseases remains unexplored. In this study, we compared the gut bacterial community of mice with wide-ranging microbial exposure to determine if low abundance bacteria vary based on microbial exposure or remain consistent. We noted that similar to the high abundance bacterial community, a core community of low abundance bacteria made up a significant portion of the gut microbiome irrespective of microbial exposure. To determine the role of low abundance bacteria in regulating community composition and host gene expression, we devised a microbiome dilution strategy to "delete" out low abundance bacteria and engrafted the diluted microbiomes into germ-free mice. Our approach successfully excluded low abundance bacteria from small and large intestinal bacterial communities and induced global changes in microbial community composition in the large intestine. Gene expression analysis of intestinal tissue revealed that loss of low abundance bacteria resulted in a drastic reduction in expression of multiple genes involved MHCII antigen presentation pathway and T-cell cytokine production in the small intestine. The effect of low abundance bacteria on MHCII expression was found to be specific to the intestinal epithelium at an early timepoint post-colonization and correlated with bacteria belonging to the family Erysipelotrichaceae. We conclude that low abundance bacteria have a significantly higher immuno-stimulatory effect compared to dominant bacteria and are thus potent drivers of early immune education in the gut.

Spatial analysis of gut microbiome reveals a distinct ecological niche associated with the mucus layer.

Mucus-associated bacterial communities are critical for determining disease pathology and promoting colonization resistance. Yet the key ecological properties of mucus resident communities remain poorly defined. Using an approach that combines in situ hybridization, laser microdissection and 16s rRNA sequencing of spatially distinct regions of the mouse gut lumen, we discovered that a dense microbial community resembling a biofilm is embedded in the mucus layer. The mucus-associated biofilm-like community excluded bacteria belonging to phylum Proteobacteria. Additionally, it was significantly more diverse and consisted of bacterial species that were unique to it. By employing germ-free mice deficient in T and B lymphocytes we found that formation of biofilm-like structure was independent of adaptive immunity. Instead the integrity of biofilm-like community depended on Gram-positive commensals such as Clostridia. Additionally, biofilm-like community in the mucus lost fewer Clostridia and showed smaller bloom of Proteobacteria compared to the lumen upon antibiotic treatment. When subjected to time-restricted feeding biofilm-like structure significantly enhanced in size and showed enrichment of Clostridia. Taken together our work discloses that mucus-associated biofilm-like community represents a specialized community that is structurally and compositionally distinct that excludes aerobic bacteria while enriching for anaerobic bacteria such as Clostridia, exhibits enhanced stability to antibiotic treatment and that can be modulated by dietary changes.

Epithelium intrinsic vitamin A signaling co-ordinates pathogen clearance in the gut via IL-18.

Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to protect against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stopΔIEC) showed higher Salmonella burden in colonic tissues early in the infection that associated with higher luminal and systemic loads of the pathogen at later stages. Higher pathogen burden in stopΔIEC mice correlated with attenuated mucosal interferon gamma (IFNγ) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stopΔIEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFNγ. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stopΔIEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFNγ production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs.

Commensals Suppress Intestinal Epithelial Cell Retinoic Acid Synthesis to Regulate Interleukin-22 Activity and Prevent Microbial Dysbiosis.

Retinoic acid (RA), a vitamin A metabolite, regulates transcriptional programs that drive protective or pathogenic immune responses in the intestine, in a manner dependent on RA concentration. Vitamin A is obtained from diet and is metabolized by intestinal epithelial cells (IECs), which operate in intimate association with microbes and immune cells. Here we found that commensal bacteria belonging to class Clostridia modulate RA concentration in the gut by suppressing the expression of retinol dehydrogenase 7 (Rdh7) in IECs. Rdh7 expression and associated RA amounts were lower in the intestinal tissue of conventional mice, as compared to germ-free mice. Deletion of Rdh7 in IECs diminished RA signaling in immune cells, reduced the IL-22-dependent antimicrobial response, and enhanced resistance to colonization by Salmonella Typhimurium. Our findings define a regulatory circuit wherein bacterial regulation of IEC-intrinsic RA synthesis protects microbial communities in the gut from excessive immune activity, achieving a balance that prevents colonization by enteric pathogens.

Autophagy: Suicide Prevention Hotline for the Gut Epithelium.

Autophagy is genetically associated with inflammatory bowel disease (IBD); however, its role remains unclear in disease pathogenesis. Three recent studies reveal a novel cytoprotective role of autophagy during viral, bacterial, and protozoan-triggered IBD (Burger et al., 2018; Matsuzawa-Ishimoto et al., 2017; Pott et al., 2018).

Alcohol Lowers Your (Intestinal) Inhibitions.

Alcohol causes microbiota dysbiosis and breaches intestinal integrity, resulting in liver inflammation and ultimately cirrhosis. In this issue of Cell Host & Microbe, Wang et al. (2016) demonstrate that ethanol suppresses the intestinal anti-microbial response. This enables gut bacteria to trespass to the liver and thus exacerbates the disease progression.

The Intestinal Mucus Layer Comes of Age.

The mucus layer is critical in limiting contact between host and the complex bacterial consortia that colonize the intestine. A recent paper in Cell Host and Microbe provides comprehensive insight into the dynamics of mucus layer maturation upon bacterial colonization of germ-free (GF) mice that have implications for studies on host-microbe interaction involving colonization of GF mice.

Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection.

Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection.

Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells.

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that are linked to immunopathology. Here we show that IFN-γ production by CD4(+) T(H)1 cells during mucosal responses to the protozoan parasite Toxoplasma gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were required for the parasite-induced intestinal pathology. The investigation of cell type-specific factors regulating T(H)1 polarization during T. gondii infection identified the T cell-intrinsic TLR pathway as a major regulator of IFN-γ production in CD4(+) T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.

The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii.

The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritance remain largely unstudied. Here we genetically characterize a nucleus-encoded homolog to the bacterial HU protein in Toxoplasma gondii. In bacteria, HU is a DNA-binding structural protein with fundamental roles in transcription, replication initiation, and DNA repair. Immunofluorescence assays reveal that in T. gondii this protein localizes to the apicoplast. We have found that the HU protein from Toxoplasma can successfully complement bacterial ΔhupA mutants, supporting a similar function. We were able to construct a genetic knockout of HU in Toxoplasma. This Δhu mutant is barely viable and exhibits significant growth retardation. Upon further analysis of the mutant phenotype, we find that this mutant has a dramatically reduced apicoplast genome copy number and, furthermore, suffers defects in the segregation of the apicoplast organelle. Our findings not only show that the HU protein is important for Toxoplasma cell biology but also demonstrate the importance of the apicoplast genome in the biogenesis of the organelle.

The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.

The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Here, we show that RegIIIγ, a secreted antibacterial lectin, is essential for maintaining a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIIIγ is a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host.

Gammadelta intraepithelial lymphocytes are essential mediators of host-microbial homeostasis at the intestinal mucosal surface.

The mammalian gastrointestinal tract harbors thousands of bacterial species that include symbionts as well as potential pathogens. The immune responses that limit access of these bacteria to underlying tissue remain poorly defined. Here we show that γδ intraepithelial lymphocytes (γδ IEL) of the small intestine produce innate antimicrobial factors in response to resident bacterial "pathobionts" that penetrate the intestinal epithelium. γδ IEL activation was dependent on epithelial cell-intrinsic MyD88, suggesting that epithelial cells supply microbe-dependent cues to γδ IEL. Finally, γδ T cells protect against invasion of intestinal tissues by resident bacteria specifically during the first few hours after bacterial encounter, indicating that γδ IEL occupy a unique temporal niche among intestinal immune defenses. Thus, γδ IEL detect the presence of invading bacteria through cross-talk with neighboring epithelial cells and are an essential component of the hierarchy of immune defenses that maintain homeostasis with the intestinal microbiota.

Eat your carrots! T cells are RARing to go.

In this issue of Immunity, Hall et al. (2011) show that vitamin A and its metabolites play a central role in regulating adaptive immunity by promoting the development of both inflammatory and regulatory T cell responses.