Designing microbial communities to starve out invading pathogens.

Identifying key features required for specific community-level functions can be challenging, especially considering the complexity of the gut microbiome. In a recent study in Science, Spragge et al. present a high-throughput experimental framework to rationally design microbial communities that can resist invasion by specific bacterial pathogens.

Biosynthetic Enzyme-guided Disease Correlation Connects Gut Microbial Metabolites Sulfonolipids to Inflammatory Bowel Disease Involving TLR4 Signaling.

The trillions of microorganisms inhabiting the human gut are intricately linked to human health. At the species abundance level, correlational studies have connected specific bacterial taxa to various diseases. While the abundances of these bacteria in the gut serve as good indicators for disease progression, understanding the functional metabolites they produce is critical to decipher how these microbes influence human health. Here, we report a unique biosynthetic enzyme-guided disease correlation approach to uncover microbial functional metabolites as potential molecular mechanisms in human health. We directly connect the expression of gut microbial sulfonolipid (SoL) biosynthetic enzymes to inflammatory bowel disease (IBD) in patients, revealing a negative correlation. This correlation is then corroborated by targeted metabolomics, identifying that SoLs abundance is significantly decreased in IBD patient samples. We experimentally validate our analysis in a mouse model of IBD, showing that SoLs production is indeed decreased while inflammatory markers are increased in diseased mice. In support of this connection, we apply bioactive molecular networking to show that SoLs consistently contribute to the immunoregulatory activity of SoL-producing human microbes. We further reveal that sulfobacins A and B, two representative SoLs, primarily target Toll-like receptor 4 (TLR4) to mediate immunomodulatory activity through blocking TLR4's natural ligand lipopolysaccharide (LPS) binding to myeloid differentiation factor 2, leading to significant suppression of LPS-induced inflammation and macrophage M1 polarization. Together, these results suggest that SoLs mediate a protective effect against IBD through TLR4 signaling and showcase a widely applicable biosynthetic enzyme-guided disease correlation approach to directly link the biosynthesis of gut microbial functional metabolites to human health.

Emerging mechanisms by which endocannabinoids and their derivatives modulate bacterial populations within the gut microbiome.

Bioactive lipids such as endocannabinoids serve as important modulators of host health and disease through their effects on various host functions including central metabolism, gut physiology, and immunity. Furthermore, changes to the gut microbiome caused by external factors such as diet or by disease development have been associated with altered endocannabinoid tone and disease outcomes. These observations suggest the existence of reciprocal relationships between host lipid signaling networks and bacterial populations that reside within the gut. Indeed, endocannabinoids and their congeners such as N-acylethanolamides have been recently shown to alter bacterial growth, functions, physiology, and behaviors, therefore introducing putative mechanisms by which these bioactive lipids directly modulate the gut microbiome. Moreover, these potential interactions add another layer of complexity to the regulation of host health and disease pathogenesis that may be mediated by endocannabinoids and their derivatives. This mini review will summarize recent literature that exemplifies how N-acylethanolamides and monoacylglycerols including endocannabinoids can impact bacterial populations in vitro and within the gut microbiome. We also highlight exciting preclinical studies that have engineered gut bacteria to synthesize host N-acylethanolamides or their precursors as potential strategies to treat diseases that are in part driven by aberrant lipid signaling, including obesity and inflammatory bowel diseases.

Agr2-associated ER stress promotes adherent-invasive E. coli dysbiosis and triggers CD103+ dendritic cell IL-23-dependent ileocolitis.

Endoplasmic reticulum (ER) stress is associated with Crohn's disease (CD), but its impact on host-microbe interaction in disease pathogenesis is not well defined. Functional deficiency in the protein disulfide isomerase anterior gradient 2 (AGR2) has been linked with CD and leads to epithelial cell ER stress and ileocolitis in mice and humans. Here, we show that ileal expression of AGR2 correlates with mucosal Enterobactericeae abundance in human inflammatory bowel disease (IBD) and that Agr2 deletion leads to ER-stress-dependent expansion of mucosal-associated adherent-invasive Escherichia coli (AIEC), which drives Th17 cell ileocolitis in mice. Mechanistically, our data reveal that AIEC-induced epithelial cell ER stress triggers CD103+ dendritic cell production of interleukin-23 (IL-23) and that IL-23R is required for ileocolitis in Agr2-/- mice. Overall, these data reveal a specific and reciprocal interaction of the expansion of the CD pathobiont AIEC with ER-stress-associated ileocolitis and highlight a distinct cellular mechanism for IL-23-dependent ileocolitis.

Long Chain Fatty Acids and Virulence Repression in Intestinal Bacterial Pathogens.

When bacterial pathogens enter the gut, they encounter a complex milieu of signaling molecules and metabolites produced by host and microbial cells or derived from external sources such as the diet. This metabolomic landscape varies throughout the gut, thus establishing a biogeographical gradient of signals that may be sensed by pathogens and resident bacteria alike. Enteric bacterial pathogens have evolved elaborate mechanisms to appropriately regulate their virulence programs, which involves sensing and responding to many of these gut metabolites to facilitate successful gut colonization. Long chain fatty acids (LCFAs) represent major constituents of the gut metabolome that can impact bacterial functions. LCFAs serve as important nutrient sources for all cellular organisms and can function as signaling molecules that regulate bacterial metabolism, physiology, and behaviors. Moreover, in several enteric pathogens, including Salmonella enterica, Listeria monocytogenes, Vibrio cholerae, and enterohemorrhagic Escherichia coli, LCFA sensing results in the transcriptional repression of virulence through two general mechanisms. First, some LCFAs function as allosteric inhibitors that decrease the DNA binding affinities of transcriptional activators of virulence genes. Second, some LCFAs also modulate the activation of histidine kinase receptors, which alters downstream intracellular signaling networks to repress virulence. This mini-review will summarize recent studies that have investigated the molecular mechanisms by which different LCFA derivatives modulate the virulence of enteric pathogens, while also highlighting important gaps in the field regarding the roles of LCFAs as determinants of infection and disease.

The Canonical Long-Chain Fatty Acid Sensing Machinery Processes Arachidonic Acid To Inhibit Virulence in Enterohemorrhagic Escherichia coli.

The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.IMPORTANCE Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. Our work introduces a defined host-pathogen interaction by which arachidonic acid, a host-derived and dietary PUFA, can impact the outcome of enteric infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC). We show that long-chain fatty acids including arachidonic acid act as signaling molecules that directly suppress a key pathogenicity island in EHEC following recognition by the fatty acyl-CoA-responsive transcription factor FadR. Thus, in addition to its established effects on host immunity and its bactericidal activities against other pathogens, we demonstrate that arachidonic acid also acts as a signaling molecule that inhibits virulence in an enteric pathogen.

Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

Bacterial signaling as an antimicrobial target.

Antibiotics profoundly reduced worldwide mortality. However, the emergence of resistance to the growth inhibiting effects of these drugs occurred. New approaches to treat infectious disease that reduce the likelihood for resistance are needed. In bacterial pathogens, complex signaling networks regulate virulence. Anti-virulence therapies aim to disrupt these networks to attenuate virulence without affecting growth. Quorum-sensing, a cell-to-cell communication system, represents an attractive anti-virulence target because it often activates virulence. The challenge is to identify druggable targets that inhibit virulence, while also minimizing the likelihood of mutations promoting resistance. Moreover, given the ubiquity of quorum-sensing systems in commensals, any potential effects of anti-virulence therapies on microbiome function should also be considered. Here we highlight the efficacy and drawbacks of anti-virulence approaches.

Dietary iron variably modulates assembly of the intestinal microbiota in colitis-resistant and colitis-susceptible mice.

Iron deficiency, a common comorbidity of gastrointestinal inflammatory disorders such as inflammatory bowel diseases (IBD), is often treated with oral iron supplementation. However, the safety of oral iron supplementation remains controversial because of its association with exacerbated disease activity in a subset of IBD patients. Because iron modulates bacterial growth and function, one possible mechanism by which iron may exacerbate inflammation in susceptible hosts is by modulating the intestinal microbiota. We, therefore, investigated the impact of dietary iron on the intestinal microbiota, utilizing the conventionalization of germ-free mice as a model of a microbial community in compositional flux to recapitulate the instability of the IBD-associated intestinal microbiota. Our findings demonstrate that altering intestinal iron availability during community assembly modulated the microbiota in non-inflamed wild type (WT) and colitis-susceptible interleukin-10-deficient (Il10-/-) mice. Depletion of luminal iron availability promoted luminal compositional changes associated with dysbiotic states irrespective of host genotype, including an expansion of Enterobacteriaceae such as Escherichia coli. Mechanistic in vitro growth competitions confirmed that high-affinity iron acquisition systems in E. coli enhance its abundance over other bacteria in iron-restricted conditions, thereby enabling pathobiont iron scavenging during dietary iron restriction. In contrast, distinct luminal community assembly was observed with dietary iron supplementation in WT versus Il10-/- mice, suggesting that the effects of increased iron on the microbiota differ with host inflammation status. Taken together, shifts in dietary iron intake during community assembly modulate the ecological structure of the intestinal microbiota and is dependent on host genotype and inflammation status.

Diet-derived galacturonic acid regulates virulence and intestinal colonization in enterohaemorrhagic Escherichia coli and Citrobacter rodentium.

Enteric pathogens sense the complex chemistry within the gastrointestinal tract to efficiently compete with the resident microbiota and establish a colonization niche. Here, we show that enterohaemorrhagic Escherichia coli and Citrobacter rodentium, its surrogate in a mouse infection model, sense galacturonic acid to initiate a multi-layered program towards successful mammalian infection. Galacturonic acid utilization as a carbon source aids the initial pathogen expansion. The main source of galacturonic acid is dietary pectin, which is converted to galacturonic acid by the prominent member of the microbiota, Bacteroides thetaiotamicron. This is regulated by the ExuR transcription factor. However, galacturonic acid is also sensed as a signal through ExuR to modulate the expression of the genes encoding a molecular syringe known as a type III secretion system, leading to infectious colitis and inflammation. Galacturonic acid acts as both a nutrient and a signal directing the exquisite microbiota-pathogen relationships within the gastrointestinal tract. This work highlights that differential dietary sugar availability influences the relationship between the microbiota and enteric pathogens, as well as disease outcomes.

Yersiniabactin-Producing Adherent/Invasive Escherichia coli Promotes Inflammation-Associated Fibrosis in Gnotobiotic Il10-/- Mice.

Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn's disease (CD). Mechanisms that favor fibrosis are not well understood, and therapeutic strategies are limited. Here we demonstrate that colitis-susceptible Il10-deficient mice develop inflammation-associated fibrosis when monoassociated with adherent/invasive Escherichia coli (AIEC) that harbors the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. fyuA-deficient AIEC also exhibited greater localization within subepithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis and corresponded with increased fibroblast activation in vitro Together, these findings suggest that Ybt establishes a profibrotic environment in the host in the absence of binding to its cognate receptor and indicate a direct link between intestinal AIEC and the induction of inflammation-associated fibrosis.

Taming the Beast: Interplay between Gut Small Molecules and Enteric Pathogens.

The overuse of antibiotics has led to the evolution of drug-resistant bacteria that are becoming increasingly dangerous to human health. According to the Centers for Disease Control and Prevention, antibiotic-resistant bacteria cause at least 2 million illnesses and 23,000 deaths in the United States annually. Traditionally, antibiotics are bactericidal or bacteriostatic agents that place selective pressure on bacteria, leading to the expansion of antibiotic-resistant strains. In addition, antibiotics that are effective against some pathogens can also exacerbate their pathogenesis and may lead to severe progression of the disease. Therefore, alternative strategies are needed to treat antibiotic-resistant bacterial infections. One novel approach is to target bacterial virulence to prevent or limit pathogen colonization, while also minimizing tissue damage and disease comorbidities in the host. This review focuses on the interactions between enteric pathogens and naturally occurring small molecules in the human gut as potential therapeutic targets for antivirulence strategies. Individual small molecules in the intestines modulate enteric pathogen virulence and subsequent intestinal fitness and colonization. Targeted interruption of pathogen sensing of these small molecules could therefore attenuate their virulence. This review highlights the paths of discovery for new classes of antimicrobials that could potentially mitigate the urgent problem of antibiotic resistance.

Intestinal bacterial biofilms modulate mucosal immune responses.

Host-associated microbial communities modulate numerous aspects of host physiology at the epithelial interface within mucosal environments. Perturbations to this symbiotic relationship between host and microbiota has been linked to numerous microbial-driven pathological states, including Crohn's disease (CD). This is in part driven by the outgrowth of aggressive resident bacterial strains such as adherent and invasive Escherichia coli (AIEC) and changes in bacterial physiology and function that promote enhanced mucosal association of pathobionts and aberrant stimulation of mucosal immunity. Endogenous bacteria from host-associated microbial communities can adopt a sessile lifestyle and form multicellular structures known as biofilms that are generated through the expression of extracellular adhesion factors that include curli amyloid fibrils, cellulose and type 1 pili. In addition to enabling bacterial attachment to mucosal surfaces, biofilm components also stimulate immune responses and can therefore instigate or perpetuate microbial-driven inflammatory diseases such as CD. These host-bacterial interactions provide pharmacological targets that can potentially be exploited to limit mucosal adherence of aggressive enteric bacteria, inappropriate stimulation of inflammatory immune responses and consequent development of chronic intestinal inflammation.

Siderophore-mediated iron acquisition and modulation of host-bacterial interactions.

Iron is an essential micronutrient for most life forms including the majority of resident bacteria of the microbiota and their mammalian hosts. Bacteria have evolved numerous mechanisms to competitively acquire iron within host environments, such as the secretion of small molecules known as siderophores that can solubilize iron for bacterial use. However, siderophore biosynthesis and acquisition is not a capability equally harbored by all resident bacteria. Moreover, the structural diversity of siderophores creates variability in the susceptibility to host mechanisms that serve to counteract siderophore-mediated iron acquisition and limit bacterial growth. As a result, the differential capabilities to acquire iron among members of a complex microbial community carry important implications for the growth and function of resident bacteria. Siderophores can also directly influence host function by modulating cellular iron homeostasis, further providing a mechanism by which resident bacteria may influence their local environment at the host-microbial interface. This review will explore the putative mechanisms by which siderophore production by resident bacteria in the intestines may influence microbial community dynamics and host-bacterial interactions with important implications for pathogen- and microbiota-driven diseases including infection, inflammatory bowel diseases and colorectal cancer.

Adherent-Invasive Escherichia coli Production of Cellulose Influences Iron-Induced Bacterial Aggregation, Phagocytosis, and Induction of Colitis.

Adherent-invasive Escherichia coli (AIEC), a functionally distinct subset of resident intestinal E. coli associated with Crohn's disease, is characterized by enhanced epithelial adhesion and invasion, survival within macrophages, and biofilm formation. Environmental factors, such as iron, modulate E. coli production of extracellular structures, which in turn influence the formation of multicellular communities, such as biofilms, and bacterial interactions with host cells. However, the physiological and functional responses of AIEC to variable iron availability have not been thoroughly investigated. We therefore characterized the impact of iron on the physiology of AIEC strain NC101 and subsequent interactions with macrophages. Iron promoted the cellulose-dependent aggregation of NC101. Bacterial cells recovered from the aggregates were more susceptible to phagocytosis than planktonic cells, which corresponded with the decreased macrophage production of the proinflammatory cytokine interleukin-12 (IL-12) p40. Prevention of aggregate formation through the disruption of cellulose production reduced the phagocytosis of iron-exposed NC101. In contrast, under iron-limiting conditions, where NC101 aggregation is not induced, the disruption of cellulose production enhanced NC101 phagocytosis and decreased macrophage secretion of IL-12 p40. Finally, abrogation of cellulose production reduced NC101 induction of colitis when NC101 was monoassociated in inflammation-prone Il10(-/-) mice. Taken together, our results introduce cellulose as a novel physiological factor that impacts host-microbe-environment interactions and alters the proinflammatory potential of AIEC.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2(-/-)) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2(-/-) mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2(-/-) mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2(-/-) mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

Altered enteric microbiota ecology in interleukin 10-deficient mice during development and progression of intestinal inflammation.

Inflammatory bowel diseases (IBD) result from dysregulated immune responses toward microbial and perhaps other luminal antigens in a genetically susceptible host, and are associated with altered composition and diversity of the intestinal microbiota. The interleukin 10-deficient (IL-10 (-/-) ) mouse has been widely used to model human IBD; however the specific alterations that occur in the intestinal microbiota of this mouse model during the onset of colonic inflammation have not yet been defined. The aim of our study was to define the changes in diversity and composition that occur in the intestinal microbiota of IL-10 (-/-) mice during the onset and progression of colonic inflammation. We used high throughput sequencing of the 16S rRNA gene to characterize the diversity and composition of formerly germ-free, wild-type and IL-10 (-/-) mice associated with the same intestinal microbiota over time. Following two weeks of colonization with a specific pathogen-free (SPF) microbiota we observed a significant increase in the diversity and richness of the intestinal microbiota of wild-type mice. In contrast, a progressive decrease in diversity and richness was observed at three and four weeks in IL-10 (-/-) mice. This decrease in diversity and richness was mirrored by an increase in Proteobacteria and Escherichia coli in IL-10 (-/-) mice. An increase in E. coli was also observed in conventionally raised IL-10 (-/-) mice at the point of colonic inflammation. Our data reports the sequential changes in diversity and composition of the intestinal microbiota in an immune-mediated mouse model that may help provide insights into the primary vs. secondary role of dysbiosis in human IBD patients.

Molecular detection of bacterial contamination in gnotobiotic rodent units.

Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.