Fusobacterium nucleatum drives a pro-inflammatory intestinal microenvironment through metabolite receptor-dependent modulation of IL-17 expression.

The colorectal cancer (CRC)-associated microbiota creates a pro-tumorigenic intestinal milieu and shapes immune responses within the tumor microenvironment. However, how oncomicrobes - like Fusobacterium nucleatum, found in the oral cavity and associated with CRC tissues- affect these distinct aspects of tumorigenesis is difficult to parse. Herein, we found that neonatal inoculation of ApcMin/+ mice with F. nucleatum strain Fn7-1 circumvents technical barriers preventing its intestinal colonization, drives colonic Il17a expression prior to tumor formation, and potentiates intestinal tumorigenesis. Using gnotobiotic mice colonized with a minimal complexity microbiota (the altered Schaedler's flora), we observed that intestinal Fn7-1 colonization increases colonic Th17 cell frequency and their IL-17A and IL-17F expression, along with a concurrent increase in colonic lamina propria Il23p19 expression. As Fn7-1 stably colonizes the intestinal tract in our models, we posited that microbial metabolites, specifically short-chain fatty acids (SCFA) that F. nucleatum abundantly produces in culture and, as we demonstrate, in the intestinal tract, might mediate part of its immunomodulatory effects in vivo. Supporting this hypothesis, we found that Fn7-1 did not alter RORγt+ CD4+T cell frequency in the absence of the SCFA receptor FFAR2. Taken together, our work suggests that F. nucleatum influences intestinal immunity by shaping Th17 responses in an FFAR2-dependent manner, although further studies are necessary to clarify the precise and multifaceted roles of FFAR2. The potential to increase intestinal Th17 responses is shared by another oncomicrobe, enterotoxigenic Bacteroides fragilis, highlighting a conserved pathway that could potentially be targeted to slow oncomicrobe-mediated CRC.

Aspirin Modulation of the Colorectal Cancer-Associated Microbe Fusobacterium nucleatum.

Aspirin is a chemopreventive agent for colorectal adenoma and cancer (CRC) that, like many drugs inclusive of chemotherapeutics, has been investigated for its effects on bacterial growth and virulence gene expression. Given the evolving recognition of the roles for bacteria in CRC, in this work, we investigate the effects of aspirin with a focus on one oncomicrobe-Fusobacterium nucleatum We show that aspirin and its primary metabolite salicylic acid alter F. nucleatum strain Fn7-1 growth in culture and that aspirin can effectively kill both actively growing and stationary Fn7-1. We also demonstrate that, at levels that do not inhibit growth, aspirin influences Fn7-1 gene expression. To assess whether aspirin modulation of F. nucleatum may be relevant in vivo, we use the ApcMin/+ mouse intestinal tumor model in which Fn7-1 is orally inoculated daily to reveal that aspirin-supplemented chow is sufficient to inhibit F. nucleatum-potentiated colonic tumorigenesis. We expand our characterization of aspirin sensitivity across other F. nucleatum strains, including those isolated from human CRC tissues, as well as other CRC-associated microbes, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli Finally, we determine that individuals who use aspirin daily have lower fusobacterial abundance in colon adenoma tissues, as determined by quantitative PCR performed on adenoma DNA. Together, our data support that aspirin has direct antibiotic activity against F. nucleatum strains and suggest that consideration of the potential effects of aspirin on the microbiome holds promise in optimizing risk-benefit assessments for use of aspirin in CRC prevention and management.IMPORTANCE There is an increasing understanding of the clinical correlations and potential mechanistic roles of specific members of the gut and tumoral microbiota in colorectal cancer (CRC) initiation, progression, and survival. However, we have yet to parlay this knowledge into better CRC outcomes through microbially informed diagnostic, preventive, or therapeutic approaches. Here, we demonstrate that aspirin, an established CRC chemopreventive, exhibits specific effects on the CRC-associated Fusobacterium nucleatum in culture, an animal model of intestinal tumorigenesis, and in human colonic adenoma tissues. Our work proposes a potential role for aspirin in influencing CRC-associated bacteria to prevent colorectal adenomas and cancer, beyond aspirin's canonical anti-inflammatory role targeting host tissues. Future research, such as studies investigating the effects of aspirin on fusobacterial load in patients, will help further elucidate the prospect of using aspirin to modulate F. nucleatumin vivo for improving CRC outcomes.

Expression of Free Fatty Acid Receptor 2 by Dendritic Cells Prevents Their Expression of Interleukin 27 and Is Required for Maintenance of Mucosal Barrier and Immune Response Against Colorectal Tumors in Mice.

BACKGROUND & AIMS: Intestinal microbes and their metabolites affect the development of colorectal cancer (CRC). Short-chain fatty acids are metabolites generated by intestinal microbes from dietary fiber. We investigated the mechanisms by which free fatty acid receptor 2 (FFAR2), a receptor for short-chain fatty acids that can affect the composition of the intestinal microbiome, contributes to the pathogenesis of CRC. METHODS: We performed studies with ApcMin/+ mice, ApcMin/+Ffar2-/- mice, mice with conditional disruption of Ffar2 in dendritic cells (DCs) (Ffar2fl/flCD11c-Cre mice), ApcMin/+Ffar2fl/flCD11c-Cre mice, and Ffar2fl/fl mice (controls); some mice were given dextran sodium sulfate to induce colitis, with or without a FFAR2 agonist or an antibody against interleukin 27 (IL27). Colon and tumor tissues were analyzed by histology, quantitative polymerase chain reaction, and 16S ribosomal RNA gene sequencing; lamina propria and mesenteric lymph node tissues were analyzed by RNA sequencing and flow cytometry. Intestinal permeability was measured after gavage with fluorescently labeled dextran. We collected data on colorectal tumors from The Cancer Genome Atlas. RESULTS: ApcMin/+Ffar2-/- mice developed significantly more spontaneous colon tumors than ApcMin/+ mice and had increased gut permeability before tumor development, associated with reduced expression of E-cadherin. Colon tumors from ApcMin/+Ffar2-/- mice had a higher number of bacteria than tumors from ApcMin/+ mice, as well as higher frequencies of CD39+CD8+ T cells and exhausted or dying T cells. DCs from ApcMin/+Ffar2-/- mice had an altered state of activation, increased death, and higher production of IL27. Administration of an antibody against IL27 reduced the numbers of colon tumors in ApcMin/+ mice with colitis. Frequencies of CD39+CD8+ T cells and IL27+ DCs were increased in colon lamina propria from Ffar2fl/flCD11c-Cre mice with colitis compared with control mice or mice without colitis. ApcMin/+Ffar2fl/flCD11c-Cre mice developed even more tumors than ApcMin/+Ffar2fl/fl mice, and their tumors had even higher numbers of IL27+ DCs. ApcMin/+ mice with colitis given the FFAR2 agonist developed fewer colon tumors, with fewer IL27+ DCs, than mice not given the agonist. DCs incubated with the FFAR2 agonist no longer had gene expression patterns associated with activation or IL27 production. CONCLUSIONS: Loss of FFAR2 promotes colon tumorigenesis in mice by reducing gut barrier integrity, increasing tumor bacterial load, promoting exhaustion of CD8+ T cells, and overactivating DCs, leading to their death. Antibodies against IL27 and an FFAR2 agonist reduce tumorigenesis in mice and might be developed for the treatment of CRC.

Insights into flagellar function and mechanism from the squid-vibrio symbiosis.

Flagella are essential and multifunctional nanomachines that not only move symbionts towards their tissue colonization site, but also play multiple roles in communicating with the host. Thus, untangling the activities of flagella in reaching, interacting, and signaling the host, as well as in biofilm formation and the establishment of a persistent colonization, is a complex problem. The squid-vibrio system offers a unique model to study the many ways that bacterial flagella can influence a beneficial association and, generally, other bacteria-host interactions. Vibrio fischeri is a bioluminescent bacterium that colonizes the Hawaiian bobtail squid, Euprymna scolopes. Over the last 15 years, the structure, assembly, and functions of V. fischeri flagella, including not only motility and chemotaxis, but also biofilm formation and symbiotic signaling, have been revealed. Here we discuss these discoveries in the perspective of other host-bacteria interactions.

The human gut bacterial genotoxin colibactin alkylates DNA.

Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.

Fusobacterium nucleatum - symbiont, opportunist and oncobacterium.

Fusobacterium nucleatum has long been found to cause opportunistic infections and has recently been implicated in colorectal cancer; however, it is a common member of the oral microbiota and can have a symbiotic relationship with its hosts. To address this dissonance, we explore the diversity and niches of fusobacteria and reconsider historic fusobacterial taxonomy in the context of current technology. We also undertake a critical reappraisal of fusobacteria with a focus on F. nucleatum as a mutualist, infectious agent and oncogenic microorganism. In this Review, we delve into recent insights and future directions for fusobacterial research, including the current genetic toolkit, our evolving understanding of its mechanistic role in promoting colorectal cancer and the challenges of developing diagnostics and therapeutics for F. nucleatum.

Gut microbiota induce IGF-1 and promote bone formation and growth.

Appreciation of the role of the gut microbiome in regulating vertebrate metabolism has exploded recently. However, the effects of gut microbiota on skeletal growth and homeostasis have only recently begun to be explored. Here, we report that colonization of sexually mature germ-free (GF) mice with conventional specific pathogen-free (SPF) gut microbiota increases both bone formation and resorption, with the net effect of colonization varying with the duration of colonization. Although colonization of adult mice acutely reduces bone mass, in long-term colonized mice, an increase in bone formation and growth plate activity predominates, resulting in equalization of bone mass and increased longitudinal and radial bone growth. Serum levels of insulin-like growth factor 1 (IGF-1), a hormone with known actions on skeletal growth, are substantially increased in response to microbial colonization, with significant increases in liver and adipose tissue IGF-1 production. Antibiotic treatment of conventional mice, in contrast, decreases serum IGF-1 and inhibits bone formation. Supplementation of antibiotic-treated mice with short-chain fatty acids (SCFAs), products of microbial metabolism, restores IGF-1 and bone mass to levels seen in nonantibiotic-treated mice. Thus, SCFA production may be one mechanism by which microbiota increase serum IGF-1. Our study demonstrates that gut microbiota provide a net anabolic stimulus to the skeleton, which is likely mediated by IGF-1. Manipulation of the microbiome or its metabolites may afford opportunities to optimize bone health and growth.

Fusobacterium nucleatum in Colorectal Carcinoma Tissue According to Tumor Location.

OBJECTIVES: Evidence suggests a possible role of Fusobacterium nucleatum in colorectal carcinogenesis, especially in right-sided proximal colorectum. Considering a change in bowel contents and microbiome from proximal to distal colorectal segments, we hypothesized that the proportion of colorectal carcinoma enriched with F. nucleatum might gradually increase along the bowel subsites from rectum to cecum. METHODS: A retrospective, cross-sectional analysis was conducted on 1,102 colon and rectal carcinomas in molecular pathological epidemiology databases of the Nurses' Health Study and the Health Professionals Follow-up Study. We measured the amount of F. nucleatum DNA in colorectal tumor tissue using a quantitative PCR assay and equally dichotomized F. nucleatum-positive cases (high vs. low). We used multivariable logistic regression analysis to examine the relationship of a bowel subsite variable (rectum, rectosigmoid junction, sigmoid colon, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon, and cecum) with the amount of F. nucleatum. RESULTS: The proportion of F. nucleatum-high colorectal cancers gradually increased from rectal cancers (2.5%; 4/157) to cecal cancers (11%; 19/178), with a statistically significant linear trend along all subsites (P<0.0001) and little evidence of non-linearity. The proportion of F. nucleatum-low cancers was higher in rectal, ascending colon, and cecal cancers than in cancers of middle segments. CONCLUSIONS: The proportion of F. nucleatum-high colorectal cancers gradually increases from rectum to cecum. Our data support the colorectal continuum model that reflects pathogenic influences of the gut microbiota on neoplastic and immune cells and challenges the prevailing two-colon (proximal vs. distal) dichotomy paradigm.

Gut Microbiota, Inflammation, and Colorectal Cancer.

Colorectal cancer is the second-leading cause of cancer-related deaths in the United States and fourth-leading cause of cancer-related deaths worldwide. While cancer is largely considered to be a disease of genetic and environmental factors, increasing evidence has demonstrated a role for the microbiota (the microorganisms associated with the human body) in shaping inflammatory environments and promoting tumor growth and spread. Herein, we discuss both human data from meta'omics analyses and data from mechanistic studies in cell culture and animal models that support specific bacterial agents as potentiators of tumorigenesis-including Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli. Further, we consider how microbes can be used in diagnosing colorectal cancer and manipulating the tumor environment to encourage better patient outcomes in response to immunotherapy treatments.

Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc.

Fusobacterium nucleatum is associated with colorectal cancer and promotes colonic tumor formation in preclinical models. However, fusobacteria are core members of the human oral microbiome and less prevalent in the healthy gut, raising questions about how fusobacteria localize to CRC. We identify a host polysaccharide and fusobacterial lectin that explicates fusobacteria abundance in CRC. Gal-GalNAc, which is overexpressed in CRC, is recognized by fusobacterial Fap2, which functions as a Gal-GalNAc lectin. F. nucleatum binding to clinical adenocarcinomas correlates with Gal-GalNAc expression and is reduced upon O-glycanase treatment. Clinical fusobacteria strains naturally lacking Fap2 or inactivated Fap2 mutants show reduced binding to Gal-GalNAc-expressing CRC cells and established CRCs in mice. Additionally, intravenously injected F. nucleatum localizes to mouse tumor tissues in a Fap2-dependent manner, suggesting that fusobacteria use a hematogenous route to reach colon adenocarcinomas. Thus, targeting F. nucleatum Fap2 or host epithelial Gal-GalNAc may reduce fusobacteria potentiation of CRC.

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide.

Bacterial flagella mediate host-microbe interactions through tissue tropism during colonization, as well as by activating immune responses. The flagellar shaft of some bacteria, including several human pathogens, is encased in a membranous sheath of unknown function. While it has been hypothesized that the sheath may allow these bacteria to evade host responses to the immunogenic flagellin subunit, this unusual structural feature has remained an enigma. Here we demonstrate that the rotation of the sheathed flagellum in both the mutualist Vibrio fischeri and the pathogen Vibrio cholerae promotes release of a potent bacteria-derived immunogen, lipopolysaccharide, found in the flagellar sheath. We further present a new role for the flagellar sheath in triggering, rather than circumventing, host immune responses in the model squid-vibrio symbiosis. Such an observation not only has implications for the study of bacterial pathogens with sheathed flagella, but also raises important biophysical questions of sheathed-flagellum function. DOI: http://dx.doi.org/10.7554/eLife.01579.001.

Genetic determinants of swimming motility in the squid light-organ symbiont Vibrio fischeri.

Bacterial flagellar motility is a complex cellular behavior required for the colonization of the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes, by the beneficial bioluminescent symbiont Vibrio fischeri. We characterized the basis of this behavior by performing (i) a forward genetic screen to identify mutants defective in soft-agar motility, as well as (ii) a transcriptional analysis to determine the genes that are expressed downstream of the flagellar master regulator FlrA. Mutants with severe defects in soft-agar motility were identified due to insertions in genes with putative roles in flagellar motility and in genes that were unexpected, including those predicted to encode hypothetical proteins and cell division-related proteins. Analysis of mutants for their ability to enter into a productive symbiosis indicated that flagellar motility mutants are deficient, while chemotaxis mutants are able to colonize a subset of juvenile squid to light-producing levels. Thirty-three genes required for normal motility in soft agar were also downregulated in the absence of FlrA, suggesting they belong to the flagellar regulon of V. fischeri. Mutagenesis of putative paralogs of the flagellar motility genes motA, motB, and fliL revealed that motA1, motB1, and both fliL1 and fliL2, but not motA2 and motB2, likely contribute to soft-agar motility. Using these complementary approaches, we have characterized the genetic basis of flagellar motility in V. fischeri and furthered our understanding of the roles of flagellar motility and chemotaxis in colonization of the juvenile squid, including identifying 11 novel mutants unable to enter into a productive light-organ symbiosis.

Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization.

Upon transit to colonization sites, bacteria often experience critical priming that prepares them for subsequent, specific interactions with the host; however, the underlying mechanisms are poorly described. During initiation of the symbiosis between the bacterium Vibrio fischeri and its squid host, which can be observed directly and in real time, approximately five V. fischeri cells aggregate along the mucociliary membranes of a superficial epithelium prior to entering host tissues. Here, we show that these few early host-associated symbionts specifically induce robust changes in host gene expression that are critical to subsequent colonization steps. This exquisitely sensitive response to the host's specific symbiotic partner includes the upregulation of a host endochitinase, whose activity hydrolyzes polymeric chitin in the mucus into chitobiose, thereby priming the symbiont and also producing a chemoattractant gradient that promotes V. fischeri migration into host tissues. Thus, the host responds transcriptionally upon initial symbiont contact, which facilitates subsequent colonization.

Chemoreceptor VfcA mediates amino acid chemotaxis in Vibrio fischeri.

Flagellar motility and chemotaxis by Vibrio fischeri are important behaviors mediating the colonization of its mutualistic host, the Hawaiian bobtail squid. However, none of the 43 putative methyl-accepting chemotaxis proteins (MCPs) encoded in the V. fischeri genome has been previously characterized. Using both an available transposon mutant collection and directed mutagenesis, we isolated mutants for 19 of these genes, and screened them for altered chemotaxis to six previously identified chemoattractants. Only one mutant was defective in responding to any of the tested compounds; the disrupted gene was thus named vfcA (Vibrio fischeri chemoreceptor A; locus tag VF_0777). In soft-agar plates, mutants disrupted in vfcA did not exhibit the serine-sensing chemotactic ring, and the pattern of migration in the mutant was not affected by the addition of exogenous serine. Using a capillary chemotaxis assay, we showed that, unlike wild-type V. fischeri, the vfcA mutant did not undergo chemotaxis toward serine and that expression of vfcA on a plasmid in the mutant was sufficient to restore the behavior. In addition to serine, we demonstrated that alanine, cysteine, and threonine are strong attractants for wild-type V. fischeri and that the attraction is also mediated by VfcA. This study thus provides the first insights into how V. fischeri integrates information from one of its 43 MCPs to respond to environmental stimuli.

Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri.

Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae ("vibrios"), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization.

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.