Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

Lipids regulate peripheral serotonin release via gut CD1d.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells.

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Testosterone: more than having the guts to win the Tour de France.

Sex bias in susceptibility to autoimmune diseases is evident but poorly characterized. Yurkovetskiy et al. (2013) report that host testosterone mediates changes in the microbiome to confer protection to adult male NOD mice from type 1 diabetes.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

Quorum Sensing Protects Pseudomonas aeruginosa against Cheating by Other Species in a Laboratory Coculture Model.

UNLABELLED: Many species of bacteria use a cell-cell communication system called quorum sensing (QS) to coordinate group activities. QS systems frequently regulate the production of exoproducts. Some of these products, such as proteases, are "public goods" that are shared among the population and vulnerable to cheating by nonproducing members of the population. Because the QS system of the opportunistic pathogen Pseudomonas aeruginosa regulates several public goods, it can serve as a model for studying cooperation. Bacteria also commonly regulate antimicrobial production through QS. In this study, we focused on the hypothesis that QS-regulated antimicrobials may be important for P. aeruginosa to protect against cheating by another bacterial species, Burkholderia multivorans. We assessed laboratory cocultures of P. aeruginosa and B. multivorans and investigated the importance of three P. aeruginosa QS-regulated antimicrobials, hydrogen cyanide, rhamnolipids, and phenazines, for competition. We found that P. aeruginosa dominates cocultures with B. multivorans and that the three antimicrobials together promote P. aeruginosa competitiveness, with hydrogen cyanide contributing the greatest effect. We show that these QS-regulated antimicrobials are also critical for P. aeruginosa to prevent B. multivorans from cheating under nutrient conditions where both species require a P. aeruginosa quorum-regulated protease for growth. Together our results highlight the importance of antimicrobials in protecting cooperating populations from exploitation by other species that can act as cheaters. IMPORTANCE: Cooperative behaviors are threatened by social cheating, wherein individuals do not produce but nonetheless benefit from shared public goods. Bacteria have been shown to use several genetic mechanisms to restrain the emergence of cheaters from within the population, but public goods might also be used by other bacterial species in the vicinity. We demonstrate that a public good produced by Pseudomonas aeruginosa can be used by another species, Burkholderia multivorans, to obtain carbon and energy. We also show that P. aeruginosa antimicrobials that are coregulated with the public good prevent invasion by the cheating species. Our results demonstrate that cross-species cheating can occur and that coregulation of public goods with antimicrobials may stabilize cooperative behavior in mixed microbial communities.

Membrane sphingolipids as essential molecular signals for Bacteroides survival in the intestine.

As predominant intestinal symbiotic bacteria, Bacteroides are essential in maintaining the health of the normal mammalian host; in return, the host provides a niche with plentiful nutrients for the symbionts. However, the intestinal environment is replete with chemical, physical, and biological challenges that require mechanisms for prompt and adept sensing of and responses to stress if the bacteria are to survive. Herein we propose that to persist in the intestine Bacteroides take advantage of their unusual bacterial sphingolipids to mediate signaling pathways previously known to be available only to higher organisms. Sphingolipids convey diverse signal transduction and stress response pathways and have profound physiological impacts demonstrated in a variety of eukaryotic cell types. We propose a mechanism by which the formation of specific sphingolipid membrane microdomains initiates signaling cascades that facilitate survival strategies within the bacteria. Our preliminary data suggest that sphingolipid signaling plays an important role in Bacteroides physiology, enabling these bacteria to persist in the intestine and to perform other functions related to symbiosis.

Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production.

BACKGROUND & AIMS: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. METHODS: We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection. RESULTS: B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. CONCLUSIONS: B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

Foxo1 controls gut homeostasis and commensalism by regulating mucus secretion.

Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

The promise and peril of transcriptional profiling in biofilm communities.

DNA microarray technology has been successfully used to identify genes that contribute to biofilm formation for a handful of bacterial species. However, as the number of profiling studies increases, it is becoming increasingly apparent that these data might miss important aspects of biofilm development. One reason for this is the inability of current experimental designs to resolve spatial and functional heterogeneity in the biofilm community. Thus, an emerging challenge is to use transcriptional profiling in combination with techniques that can identify and separate relevant subpopulations within a biofilm.

Multifunctional and recollectable carbon nanotube ponytails for water purification.

Carbon nanotubes (CNTs) are promising nanomaterials that have the potential to revolutionize water treatment practices in the future. The direct use of unbounded CNTs, however, poses health risks to humans and ecosystems because they are difficult to separate from treated water. Here, we report the design and synthesis of carbon nanotube ponytails (CNPs) by integrating CNTs into micrometer-sized colloidal particles, which greatly improves the effectiveness of post-treatment separation using gravitational sedimentation, magnetic attraction, and membrane filtration. We further demonstrate that CNPs can effectively perform major treatment tasks including adsorption, disinfection, and catalysis. Using model pollutants such as methylene blue, Escherichia coli, and p-nitrophenol, we show that all the surfaces of individual CNTs in CNPs are accessible during water treatment. Our results suggest that the rational design of hierarchical structures represents a feasible approach to develop nanomaterials for engineering applications such as water and wastewater treatment.

Microbial exposure during early life has persistent effects on natural killer T cell function.

Exposure to microbes during early childhood is associated with protection from immune-mediated diseases such as inflammatory bowel disease (IBD) and asthma. Here, we show that in germ-free (GF) mice, invariant natural killer T (iNKT) cells accumulate in the colonic lamina propria and lung, resulting in increased morbidity in models of IBD and allergic asthma as compared with that of specific pathogen-free mice. This was associated with increased intestinal and pulmonary expression of the chemokine ligand CXCL16, which was associated with increased mucosal iNKT cells. Colonization of neonatal-but not adult-GF mice with a conventional microbiota protected the animals from mucosal iNKT accumulation and related pathology. These results indicate that age-sensitive contact with commensal microbes is critical for establishing mucosal iNKT cell tolerance to later environmental exposures.

The pathogenic properties of a novel and conserved gene product, KerV, in proteobacteria.

Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design.

Quorum sensing and motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures.

In the environment, multiple microbial taxa typically coexist as communities, competing for resources and, often, physically associated within biofilms. A dual-species cocultivation model has been developed by using two ubiquitous and well studied microbes Pseudomonas aeruginosa (P.a.) and Agrobacterium tumefaciens (A.t.) as a tractable system to identify molecular mechanisms that underlie multispecies microbial associations. Several factors were found to influence coculture interactions. P.a. had a distinct growth-rate advantage in cocultures, increasing its relative abundance during planktonic and biofilm growth. P.a. also demonstrated a slight quorum-sensing-dependent increase in growth yield in liquid cocultures. P.a. dominated coculture biofilms, "blanketing" or burying immature A.t. microcolonies. P.a. flagellar and type IV pili mutant strains exhibited deficient blanketing and impaired competition in coculture biofilms, whereas, in planktonic coculture, these mutations had no effect on competition. In contrast, A.t. used motility to emigrate from coculture biofilms. In both planktonic and biofilm cocultures, A.t. remained viable for extended periods of time, coexisting with its more numerous competitor. These findings reveal that quorum-sensing-regulated functions and surface motility are important microbial competition factors for P.a. and that the outcome of competition and the relative contribution of different factors to competition are strongly influenced by the environment in which they occur.

Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people suffering from cystic fibrosis (CF). In CF airways, P. aeruginosa forms surface-associated communities called biofilms. Compared with free-swimming cultures, biofilms resist clearance by the host immune system and display increased resistance to antimicrobial agents. In this study we developed a technique to coat surfaces with molecules that are abundant in CF airways in order to investigate their impact on P. aeruginosa biofilm development. We found that P. aeruginosa biofilm development proceeds differently on surfaces coated with the glycoprotein mucin compared with biofilm development on glass and surfaces coated with actin or DNA. Biofilms formed on mucin-coated surfaces developed large cellular aggregates and had increased tolerance to the antibiotic tobramycin compared with biofilms grown on glass. Analysis of selected mutant backgrounds in conjunction with time-lapse microscopy revealed that surface-associated motility was blocked on the mucin surface. Furthermore, our data suggest that a specific adhesin-mucin interaction immobilizes the bacterium on the surface. Together, these experiments suggest that mucin, which may serve as an attachment surface in CF airways, impacts P. aeruginosa biofilm development and function.