Metabolic Interaction Between Host and the Gut Microbiota During High-Fat Diet-Induced Colorectal Cancer.

Colorectal cancer (CRC) is the second-highest cause of cancer-associated mortality among both men and women worldwide. One of the risk factors for CRC is obesity, which is correlated with a high-fat diet prevalent in Western dietary habits. The association between an obesogenic high-fat diet and CRC has been established for several decades; however, the mechanisms by which a high-fat diet increases the risk of CRC remain unclear. Recent studies indicate that gut microbiota strongly influence the pathogenesis of both high-fat diet-induced obesity and CRC. The gut microbiota is composed of hundreds of bacterial species, some of which are implicated in CRC. In particular, the expansion of facultative anaerobic Enterobacteriaceae, which is considered a microbial signature of intestinal microbiota functional imbalance (dysbiosis), is associated with both high-fat diet-induced obesity and CRC. Here, we review the interaction between the gut microbiome and its metabolic byproducts in the context of colorectal cancer (CRC) during high-fat diet-induced obesity. In addition, we will cover how a high-fat diet can drive the expansion of genotoxin-producing Escherichia coli by altering intestinal epithelial cell metabolism during gut inflammation conditions.

An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance.

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

Colonization resistance: metabolic warfare as a strategy against pathogenic Enterobacteriaceae.

The intestine is home to a large and complex bacterial ecosystem (microbiota), which performs multiple beneficial functions for the host, including immune education, nutrition, and protection against invasion by enteric pathogens (colonization resistance). The host and microbiome symbiotic interactions occur in part through metabolic crosstalk. Thus, microbiota members have evolved highly diverse metabolic pathways to inhibit pathogen colonization via activation of protective immune responses and nutrient acquisition and utilization. Conversely, pathogenic Enterobacteriaceae actively induce an inflammation-dependent disruption of the gut microbial ecosystem (dysbiosis) to gain a competitive metabolic advantage against the resident microbiota. This review discusses the recent findings on the crucial role of microbiota metabolites in colonization resistance regulation. Additionally, we summarize metabolic mechanisms used by pathogenic Enterobacteriaceae to outcompete commensal microbes and cause disease.

High-fat diet-induced colonocyte dysfunction escalates microbiota-derived trimethylamine N-oxide.

A Western-style, high-fat diet promotes cardiovascular disease, in part because it is rich in choline, which is converted to trimethylamine (TMA) by the gut microbiota. However, whether diet-induced changes in intestinal physiology can alter the metabolic capacity of the microbiota remains unknown. Using a mouse model of diet-induced obesity, we show that chronic exposure to a high-fat diet escalates Escherichia coli choline catabolism by altering intestinal epithelial physiology. A high-fat diet impaired the bioenergetics of mitochondria in the colonic epithelium to increase the luminal bioavailability of oxygen and nitrate, thereby intensifying respiration-dependent choline catabolism of E. coli In turn, E. coli choline catabolism increased levels of circulating trimethlamine N-oxide, which is a potentially harmful metabolite generated by gut microbiota.

A Nitrogen Metabolic Enzyme Provides Salmonella Fitness Advantage by Promoting Utilization of Microbiota-Derived Carbon Source.

Microbes support their growth in vertebrate hosts by exploiting a large variety of dietary components as nutrients, which determines the composition of gut microbiota. A pathogen Salmonella expands by utilizing 1,2-propanediol, a microbiota-fermented product, during mucosal inflammation. However, it remains largely unknown how the pathogen decides which nutrient to consume from the complex mixture in the gut. Here, we show that Salmonella enterica serovar Typhimurium utilizes 1,2-propanediol by EIIANtr (a nitrogen-metabolic PTS component implicated in virulence)-mediated regulation of the pdu operon, thereby expanding in the murine intestine. Propionyl-CoA, a metabolic intermediate produced by 1,2-propanediol catabolism, elevates EIIANtr protein amounts, entailing positive feedback, thereby boosting the 1,2-propanediol-utilization process. EIIANtr promotes pdu expression by enhancing glutathione synthesis. CRP (cAMP receptor protein) induces pdu genes by increasing EIIANtr expression in response to glucose availability. Notably, EIIANtr-mediated 1,2-propanediol-utilization conferred a growth benefit even under high glucose conditions which reduces CRP activity. The EIIANtr-mediated activation is likely conserved in pathogenic enterobacteria including Escherichia coli. Collectively, our findings suggest that Salmonella promotes its fitness by precisely modulating the utilization system for microbiota-derived carbon source. They also suggest that Salmonella may integrate signals, processed via EIIANtr, into its metabolic program as well as virulence circuit.

Programmed Delay of a Virulence Circuit Promotes Salmonella Pathogenicity.

Signal transduction systems dictate various cellular behaviors in response to environmental changes. To operate cellular programs appropriately, organisms have sophisticated regulatory factors to optimize the signal response. The PhoP/PhoQ master virulence regulatory system of the intracellular pathogen Salmonella enterica is activated inside acidic macrophage phagosomes. Here we report that Salmonella delays the activation of this system inside macrophages using an inhibitory protein, EIIANtr (a component of the nitrogen-metabolic phosphotransferase system). We establish that EIIANtr directly restrains PhoP binding to its target promoter, thereby negatively controlling the expression of PhoP-activated genes. PhoP furthers its activation by promoting Lon-mediated degradation of EIIANtr at acidic pH. These results suggest that Salmonella ensures robust activation of its virulence system by suspending the activation of PhoP until a sufficient level of active PhoP is present to overcome the inhibitory effect of EIIANtr Our findings reveal how a pathogen precisely and efficiently operates its virulence program during infection.IMPORTANCE To accomplish successful infection, pathogens must operate their virulence programs in a precise, time-sensitive, and coordinated manner. A major question is how pathogens control the timing of virulence gene expression during infection. Here we report that the intracellular pathogen Salmonella controls the timing and level of virulence gene expression by using an inhibitory protein, EIIANtr A DNA binding master virulence regulator, PhoP, controls various virulence genes inside acidic phagosomes. Salmonella decreases EIIANtr amounts at acidic pH in a Lon- and PhoP-dependent manner. This, in turn, promotes expression of the PhoP-activated virulence program because EIIANtr hampers activation of PhoP-regulated genes by interfering with PhoP binding to DNA. EIIANtr enables Salmonella to impede the activation of PhoP-regulated gene expression inside macrophages. Our findings suggest that Salmonella achieves programmed delay of virulence gene activation by adjusting levels of an inhibitory factor.

Genetic Ablation of Butyrate Utilization Attenuates Gastrointestinal Salmonella Disease.

Salmonella enterica serovar (S.) Typhi is an extraintestinal pathogen that evolved from Salmonella serovars causing gastrointestinal disease. Compared with non-typhoidal Salmonella serovars, the genomes of typhoidal serovars contain various loss-of-function mutations. However, the contribution of these genetic differences to this shift in pathogen ecology remains unknown. We show that the ydiQRSTD operon, which is deleted in S. Typhi, enables S. Typhimurium to utilize microbiota-derived butyrate during gastrointestinal disease. Unexpectedly, genetic ablation of butyrate utilization reduces S. Typhimurium epithelial invasion and attenuates intestinal inflammation. Deletion of ydiD renders S. Typhimurium sensitive to butyrate-mediated repression of invasion gene expression. Combined with the gain of virulence-associated (Vi) capsular polysaccharide and loss of very-long O-antigen chains, two features characteristic of S. Typhi, genetic ablation of butyrate utilization abrogates S. Typhimurium-induced intestinal inflammation. Thus, the transition from a gastrointestinal to an extraintestinal pathogen involved discrete genetic changes, providing insights into pathogen evolution and emergence.

The role of the FliD C-terminal domain in pentamer formation and interaction with FliT.

Flagellar biogenesis is controlled by a negative feedback loop. When FliD was secreted at the late step of flagellar assembly, the FliD-FliT complex disassembled and free FliT bound to the FlhDC complex, a master regulator of flagellar biogenesis, subsequently inhibiting the overall expression of flagellar proteins. In this study, we analyzed the role of the FliD C-terminal domain in pentamer formation and interaction with FliT. Our study showed that the FliD L443R mutant exists as a monomer in solution, indicating that the Leu443 residue of FliD, which contributes to its interaction with FliT, plays a crucial role in the pentameric oligomerization of FliD. Consistently, the increased levels of free FliT proteins caused by FliD L443R mutation had negative effects on the gene expression of flagellar synthesis and reduced the expression of flagellar proteins. The lengths of flagella in each cell were significantly reduced in L443R mutant strain, suggesting that normal flagellar biogenesis was impeded. These results suggest that the C-terminal domain of FliD plays a crucial role in the pentameric oligmerization of FliD and the binding of FliT to the C-terminal domain of FliD is critical to inhibit the premature assembly of the FliD pentamer in the cytosol.

Enzyme IIANtr Regulates Salmonella Invasion Via 1,2-Propanediol And Propionate Catabolism.

Many Proteobacteria possess a nitrogen-metabolic phosphotransferase system (PTSNtr) consisting of EINtr, NPr, and EIIANtr (encoded by ptsP, ptsO, and ptsN, respectively). The PTSNtr plays diverse regulatory roles, but the substrate phosphorylated by EIIANtr and its primary functions have not yet been identified. To comprehensively understand the roles of PTSNtr in Salmonella Typhimurium, we compared the whole transcriptomes of wild-type and a ΔptsN mutant. Genome-wide RNA sequencing revealed that 3.5% of the annotated genes were up- or down-regulated by three-fold or more in the absence of EIIANtr. The ΔptsN mutant significantly down-regulated the expression of genes involved in vitamin B12 synthesis, 1,2-propanediol utilization, and propionate catabolism. Moreover, the invasiveness of the ΔptsN mutant increased about 5-fold when 1,2-propanediol or propionate was added, which was attributable to the increased stability of HilD, the transcriptional regulator of Salmonella pathogenicity island-1. Interestingly, an abundance of 1,2-propanediol or propionate promoted the production of EIIANtr, suggesting the possibility of a positive feedback loop between EIIANtr and two catabolic pathways. These results demonstrate that EIIANtr is a key factor for the utilization of 1,2-propanediol and propionate as carbon and energy sources, and thereby modulates the invasiveness of Salmonella via 1,2-propanediol or propionate catabolism.

Fine-tuning of amino sugar homeostasis by EIIA(Ntr) in Salmonella Typhimurium.

The nitrogen-metabolic phosphotransferase system, PTS(Ntr), consists of the enzymes I(Ntr), NPr and IIA(Ntr) that are encoded by ptsP, ptsO, and ptsN, respectively. Due to the proximity of ptsO and ptsN to rpoN, the PTS(Ntr) system has been postulated to be closely related with nitrogen metabolism. To define the correlation between PTS(Ntr) and nitrogen metabolism, we performed ligand fishing with EIIA(Ntr) as a bait and revealed that D-glucosamine-6-phosphate synthase (GlmS) directly interacted with EIIA(Ntr). GlmS, which converts D-fructose-6-phosphate (Fru6P) into D-glucosamine-6-phosphate (GlcN6P), is a key enzyme producing amino sugars through glutamine hydrolysis. Amino sugar is an essential structural building block for bacterial peptidoglycan and LPS. We further verified that EIIA(Ntr) inhibited GlmS activity by direct interaction in a phosphorylation-state-dependent manner. EIIA(Ntr) was dephosphorylated in response to excessive nitrogen sources and was rapidly degraded by Lon protease upon amino sugar depletion. The regulation of GlmS activity by EIIA(Ntr) and the modulation of glmS translation by RapZ suggest that the genes comprising the rpoN operon play a key role in maintaining amino sugar homeostasis in response to nitrogen availability and the amino sugar concentration in the bacterial cytoplasm.