Time resolved multi-omics reveals diverse metabolic strategies of Salmonella during diet-induced inflammation.

With a rise in antibiotic resistance and chronic infection, the metabolic response of Salmonella enterica serovar Typhimurium to various dietary conditions over time remains an understudied avenue for novel, targeted therapeutics. Elucidating how enteric pathogens respond to dietary variation not only helps us decipher the metabolic strategies leveraged for expansion but also assists in proposing targets for therapeutic interventions. Here, we use a multi-omics approach to identify the metabolic response of Salmonella enterica serovar Typhimurium in mice on both a fibrous diet and high-fat diet over time. When comparing Salmonella gene expression between diets, we found a preferential use of respiratory electron acceptors consistent with increased inflammation of the high-fat diet mice. Looking at the high-fat diet over the course of infection, we noticed heterogeneity of samples based on Salmonella ribosomal activity, which separated into three infection phases: early, peak, and late. We identified key respiratory, carbon, and pathogenesis gene expression descriptive of each phase. Surprisingly, we identified genes associated with host-cell entry expressed throughout infection, suggesting sub-populations of Salmonella or stress-induced dysregulation. Collectively, these results highlight not only the sensitivity of Salmonella to its environment but also identify phase-specific genes that may be used as therapeutic targets to reduce infection. Importance: Identifying novel therapeutic strategies for Salmonella infection that occur in relevant diets and over time is needed with the rise of antibiotic resistance and global shifts towards Western diets that are high in fat and low in fiber. Mice on a high-fat diet are more inflamed compared to those on a fibrous diet, creating an environment that results in more favorable energy generation for Salmonella . Over time on a high-fat diet, we observed differential gene expression across infection phases. Together, these findings reveal the metabolic tuning of Salmonella to dietary and temporal perturbations. Research like this, exploring the dimensions of pathogen metabolic plasticity, can pave the way for rationally designed strategies to control disease.

Gut microbiome carbon and sulfur metabolisms support Salmonella during pathogen infection.

Salmonella enterica serovar Typhimurium is a pervasive enteric pathogen and an ongoing global threat to public health. Ecological studies in the Salmonella impacted gut remain underrepresented in the literature, discounting the microbiome mediated interactions that may inform Salmonella physiology during colonization and infection. To understand the microbial ecology of Salmonella remodeling of the gut microbiome, here we performed multi-omics approaches on fecal microbial communities from untreated and Salmonella -infected mice. Reconstructed genomes recruited metatranscriptomic and metabolomic data providing a strain-resolved view of the expressed metabolisms of the microbiome during Salmonella infection. This data informed possible Salmonella interactions with members of the gut microbiome that were previously uncharacterized. Salmonella- induced inflammation significantly reduced the diversity of transcriptionally active members in the gut microbiome, yet increased gene expression was detected for 7 members, with Luxibacter and Ligilactobacillus being the most active. Metatranscriptomic insights from Salmonella and other persistent taxa in the inflamed microbiome further expounded the necessity for oxidative tolerance mechanisms to endure the host inflammatory responses to infection. In the inflamed gut lactate was a key metabolite, with microbiota production and consumption reported amongst transcriptionally active members. We also showed that organic sulfur sources could be converted by gut microbiota to yield inorganic sulfur pools that become oxidized in the inflamed gut, resulting in thiosulfate and tetrathionate that supports Salmonella respiration. Advancement of pathobiome understanding beyond inferences from prior amplicon-based approaches can hold promise for infection mitigation, with the active community outlined here offering intriguing organismal and metabolic therapeutic targets.

Exposing new taxonomic variation with inflammation - a murine model-specific genome database for gut microbiome researchers.

BACKGROUND: The murine CBA/J mouse model widely supports immunology and enteric pathogen research. This model has illuminated Salmonella interactions with the gut microbiome since pathogen proliferation does not require disruptive pretreatment of the native microbiota, nor does it become systemic, thereby representing an analog to gastroenteritis disease progression in humans. Despite the value to broad research communities, microbiota in CBA/J mice are not represented in current murine microbiome genome catalogs. RESULTS: Here we present the first microbial and viral genomic catalog of the CBA/J murine gut microbiome. Using fecal microbial communities from untreated and Salmonella-infected, highly inflamed mice, we performed genomic reconstruction to determine the impacts on gut microbiome membership and functional potential. From high depth whole community sequencing (~ 42.4 Gbps/sample), we reconstructed 2281 bacterial and 4516 viral draft genomes. Salmonella challenge significantly altered gut membership in CBA/J mice, revealing 30 genera and 98 species that were conditionally rare and unsampled in non-inflamed mice. Additionally, inflamed communities were depleted in microbial genes that modulate host anti-inflammatory pathways and enriched in genes for respiratory energy generation. Our findings suggest decreases in butyrate concentrations during Salmonella infection corresponded to reductions in the relative abundance in members of the Alistipes. Strain-level comparison of CBA/J microbial genomes to prominent murine gut microbiome databases identified newly sampled lineages in this resource, while comparisons to human gut microbiomes extended the host relevance of dominant CBA/J inflammation-resistant strains. CONCLUSIONS: This CBA/J microbiome database provides the first genomic sampling of relevant, uncultivated microorganisms within the gut from this widely used laboratory model. Using this resource, we curated a functional, strain-resolved view on how Salmonella remodels intact murine gut communities, advancing pathobiome understanding beyond inferences from prior amplicon-based approaches. Salmonella-induced inflammation suppressed Alistipes and other dominant members, while rarer commensals like Lactobacillus and Enterococcus endure. The rare and novel species sampled across this inflammation gradient advance the utility of this microbiome resource to benefit the broad research needs of the CBA/J scientific community, and those using murine models for understanding the impact of inflammation on the gut microbiome more generally. Video Abstract.

Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay.

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 µM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki' (inhibitor constant) values ranging from 26 to 116 µM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections.

Sugar-Phosphate Toxicities Attenuate Salmonella Fitness in the Gut.

Pathogens are becoming resistant to antimicrobials at an increasing rate, and novel therapeutic strategies are needed. Using Salmonella as a model, we have investigated the induction of sugar-phosphate toxicity as a potential therapeutic modality. The approach entails providing a nutrient while blocking the catabolism of that nutrient, resulting in the accumulation of a toxic intermediate. We hypothesize that this build-up will decrease the fitness of the organism during infection given nutrient availability. We tested this hypothesis using mutants lacking one of seven genes whose mutation is expected to cause the accumulation of a toxic metabolic intermediate. The araD, galE, rhaD, glpD, mtlD, manA, and galT mutants were then provided the appropriate sugars, either in vitro or during gastrointestinal infection of mice. All but the glpD mutant had nutrient-dependent growth defects in vitro, suggestive of sugar-phosphate toxicity. During gastrointestinal infection of mice, five mutants had decreased fitness. Providing the appropriate nutrient in the animal's drinking water was required to cause fitness defects with the rhaD and manA mutants and to enhance the fitness defect of the araD mutant. The galE and mtlD mutants were severely attenuated regardless of the nutrient being provided in the drinking water. Homologs of galE are widespread among bacteria and in humans, rendering the specific targeting of bacterial pathogens difficult. However, the araD, mtlD, and rhaD genes are not present in humans, appear to be rare in most phyla of bacteria, and are common in several genera of Enterobacteriaceae, making the encoded enzymes potential narrow-spectrum therapeutic targets. IMPORTANCE Bacterial pathogens are becoming increasingly resistant to antibiotics. There is an urgent need to identify novel drug targets and therapeutic strategies. In this work we have assembled and characterized a collection of mutations in our model pathogen, Salmonella enterica, that block a variety of sugar utilization pathways in such a way as to cause the accumulation of a toxic sugar-phosphate. Mutations in three genes, rhaD, araD, and mtlD, dramatically decrease the fitness of Salmonella in a mouse model of gastroenteritis, suggesting that RhaD, AraD, and MtlD may be good narrow-spectrum drug targets. The induction of sugar-phosphate toxicities may be a therapeutic strategy that is broadly relevant to other bacterial and fungal pathogens.

Heating Rate during Shell Egg Thermal Treatment Elicits Stress Responses and Alters Virulence of Salmonella enterica Serovar Enteritidis; Implications for Shell Egg Pasteurization.

Thermal pasteurization of shell eggs, at various time-temperature combinations, has been proposed previously and implemented industrially. This study was conducted to determine if shell egg heating rate, which varies with different pasteurization implementations, alters the Salmonella enterica serovar Enteritidis response to different stresses or expression of virulence. Shell eggs, containing Salmonella Enteritidis in yolk, were subjected to a low (2.4°C/min) or a high (3.5°C/min) heating rate during treatments that mimicked the pasteurization temperature come-up stage. The low heating rate protected Salmonella from the following processes: (i) lethal heat at the holding stage, (ii) loss of viability during 8-h cooling after heating, and (iii) sequential antimicrobial ozone treatment. Transcriptional analysis using Salmonella reporter strains revealed that the heat stress response gene grpE was transcribed at 3-fold-higher levels (P = 0.0009) at the low than at the high heating rate. Slow heating also significantly increased the transcription of the Salmonella virulence-related genes sopB (P = 0.0012) and sseA (P = 0.0006) in comparison to fast heating. Salmonella virulence was determined experimentally as 50% lethal dose (LD50) values in an in vivo model. The slow heat treatment mildly increased Salmonella Enteritidis virulence in mice (LD50 of 3.3 log CFU), compared to that in nontreated yolk (LD50 of 3.9 log CFU). However, when ozone application followed the slow heat treatment, Salmonella virulence decreased (LD50 of 4.2 log CFU) compared to that for heat-treated or nontreated yolk. In conclusion, heating shell eggs at a low rate can trigger hazardous responses that may compromise the safety of the final pasteurized products but following the thermal treatment with ozone application may help alleviate these concerns. IMPORTANCE Pasteurization of shell eggs is an important technology designed to protect consumers against Salmonella Enteritidis that contaminates this commodity. A low heating rate is preferred over a high rate during shell egg thermal pasteurization due to product quality concern. However, it is not known whether raising the temperature at different rates, during pasteurizing, would potentially affect product safety determinants. The current study demonstrated that slow heating during the pasteurization come-up stage increased the following risks: (i) resistance of Salmonella to pasteurization holding stage or to subsequent ozone treatment, (ii) recovery of Salmonella during the cooling that followed pasteurization, and (iii) Salmonella's ability to cause disease (i.e., virulence). Our findings inform food processors about potential safety risks to consumers resulting from improper use of processing parameters during shell egg pasteurization. Additionally, treating shell eggs with ozone after heat treatment could alleviate these hazards and protect consumers from natural Salmonella Enteritidis contaminants in shell eggs.

Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 µM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

The Role of Egg Yolk in Modulating the Virulence of Salmonella Enterica Serovar Enteritidis.

Contribution of food vehicles to pathogenicity of disease-causing microorganisms is an important but overlooked research field. The current study was initiated to reveal the relationship between virulence of Salmonella enterica serovar Enteritidis and egg yolk as a hosting medium. Mice were orally challenged with Salmonella Enteritidis cultured in egg yolk or tryptic soy broth (TSB). Additionally, mice were challenged with Salmonella Enteritidis cultured in TSB, followed by administration of sterile egg yolk, to discern the difference between pre-growth of the pathogen and its mere presence in egg yolk during infection. The pathogen's Lethal dose 50 (LD50) was the lowest when grown in yolk (2.8×102 CFU), compared to 1.1×103 CFU in TSB, and 4.6×103 CFU in TSB followed by administration of sterile yolk. Additionally, mice that orally received Salmonella Enteritidis grown in egg yolk expressed a high death rate. These findings were supported by transcriptional analysis results. Expression of promoters of virulence-related genes (sopB and sseA) in genetically modified Salmonella Enteritidis reporter strains was significantly higher (p < 0.05) when the bacterium was grown in the yolk, compared to that grown in TSB. Sequencing of RNA (RNA-seq) revealed 204 differentially transcribed genes in Salmonella Enteritidis grown in yolk vs. TSB. Yolk-grown Salmonella Enteritidis exhibited upregulated virulence pathways, including type III secretion systems, epithelial cell invasion, and infection processes; these observations were confirmed by RT-qPCR results. The transcriptomic analysis suggested that upregulation of virulence machinery of Salmonella Enteritidis grown in egg yolk was related to increased iron uptake, biotin utilization, flagellar biosynthesis, and export of virulence proteins encoded on Salmonella pathogenicity island 1, 2, 4, and 5. These biological responses may have acted in concert to increase the virulence of Salmonella infection in mice. In conclusion, growth in egg yolk enhanced Salmonella Enteritidis virulence, indicating the significance of this food vehicle to the risk assessment of salmonellosis.

Optimization of proteomics sample preparation for identification of host and bacterial proteins in mouse feces.

Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris-HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins.

Sugar-Phosphate Toxicities.

Accumulation of phosphorylated intermediates during cellular metabolism can have wide-ranging toxic effects on many organisms, including humans and the pathogens that infect them. These toxicities can be induced by feeding an upstream metabolite (a sugar, for instance) while simultaneously blocking the appropriate metabolic pathway with either a mutation or an enzyme inhibitor. Here, we survey the toxicities that can arise in the metabolism of glucose, galactose, fructose, fructose-asparagine, glycerol, trehalose, maltose, mannose, mannitol, arabinose, and rhamnose. Select enzymes in these metabolic pathways may serve as novel therapeutic targets. Some are conserved broadly among prokaryotes and eukaryotes (e.g., glucose and galactose) and are therefore unlikely to be viable drug targets. However, others are found only in bacteria (e.g., fructose-asparagine, rhamnose, and arabinose), and one is found in fungi but not in humans (trehalose). We discuss what is known about the mechanisms of toxicity and how resistance is achieved in order to identify the prospects and challenges associated with targeted exploitation of these pervasive metabolic vulnerabilities.

Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase.

Salmonellais a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.

Role of CsrA in stress responses and metabolism important for Salmonella virulence revealed by integrated transcriptomics.

To cause infection, Salmonella must survive and replicate in host niches that present dramatically different environmental conditions. This requires a flexible metabolism and physiology, responsive to conditions of the local milieu. The sequence specific RNA binding protein CsrA serves as a global regulator that governs gene expression required for pathogenicity, metabolism, biofilm formation, and motility in response to nutritional conditions. Its activity is determined by two noncoding small RNAs (sRNA), CsrB and CsrC, which sequester and antagonize this protein. Here, we used ribosome profiling and RNA-seq analysis to comprehensively examine the effects of CsrA on mRNA occupancy with ribosomes, a measure of translation, transcript stability, and the steady state levels of transcripts under in vitro SPI-1 inducing conditions, to simulate growth in the intestinal lumen, and under in vitro SPI-2-inducing conditions, to simulate growth in the Salmonella containing vacuole (SCV) of the macrophage. Our findings uncovered new roles for CsrA in controlling the expression of structural and regulatory genes involved in stress responses, metabolism, and virulence systems required for infection. We observed substantial variation in the CsrA regulon under the two growth conditions. In addition, CsrB/C sRNA levels were greatly reduced under the simulated intracellular conditions and were responsive to nutritional factors that distinguish the intracellular and luminal environments. Altogether, our results reveal CsrA to be a flexible regulator, which is inferred to be intimately involved in maintaining the distinct gene expression patterns associated with growth in the intestine and the macrophage.

Salmonella-Mediated Inflammation Eliminates Competitors for Fructose-Asparagine in the Gut.

Salmonella enterica elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent upon Salmonella pathogenicity islands 1 and 2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg (dry weight). F-Asn is also present in the intestinal tract of germfree mice at 2,700 pmol/mg (dry weight) and in the intestinal tract of conventional mice at 9 to 28 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class Clostridia encode F-Asn utilization pathways and that they are eliminated from highly inflamed Salmonella-infected mice. Collectively, our studies identify the source of F-Asn as the diet and that Salmonella-mediated inflammation is required to eliminate competitors and allow the pathogen nearly exclusive access to this nutrient.

Identification of Bacterial Species That Can Utilize Fructose-Asparagine.

Salmonella enterica serovar Typhimurium is the only organism demonstrated to utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. In this report, we first used a bioinformatics approach to identify other microorganisms that encode homologs of the Salmonella F-Asn utilization enzymes FraB (deglycase), FraD (kinase), and FraE (asparaginase). These candidate organisms were then tested with up to four different methods to confirm their ability to utilize F-Asn. The easiest and most broadly applicable method utilized a biological toxicity assay, which is based on the observation that F-Asn is toxic to a Salmonella fraB mutant. Candidate organisms were grown in a rich medium containing F-Asn, and depletion of F-Asn from the medium was inferred by the growth of a Salmonella fraB mutant in that same medium. For select organisms, the toxicity assay was cross-validated by direct mass spectrometry-aided measurement of F-Asn in the spent-culture media and through demonstration of FraB and FraD enzyme activity in cellular extracts. For prototrophs, F-Asn utilization was additionally confirmed by growth in a minimal medium containing F-Asn as the sole carbon source. Collectively, these studies established that Clostridiumbolteae, Clostridium acetobutylicum, and Clostridium clostridioforme can utilize F-Asn, but Clostridium difficile cannot; Klebsiella oxytoca and some Klebsiella pneumoniae subspecies can utilize F-Asn; and some Citrobacter rodentium and Citrobacter freundii strains can also utilize F-Asn. Within Salmonella enterica, the host-adapted serovars Typhi and Paratyphi A have lost the ability to utilize F-Asn.IMPORTANCE Fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and it is also a nutrient source for Salmonella enterica, a foodborne pathogen. Here, we determined that among the normal intestinal microbiota, there are species of Clostridium that encode the enzymes required for F-Asn utilization. Using complementary experimental approaches, we have confirmed that three members of Clostridium, two members of Klebsiella, and two members of Citrobacter can indeed utilize F-Asn. The Clostridium spp. likely compete with Salmonella for F-Asn in the gut and contribute to competitive exclusion. FraB, one of the enzymes in the F-Asn utilization pathway, is a potential drug target because inhibition of this enzyme leads to the accumulation of a toxic metabolite that inhibits the growth of Salmonella species. This study identifies the potential off-target organisms that need to be considered when developing therapeutics directed at FraB.

Methods to Study Solo/Orphan Quorum-Sensing Receptors.

LuxR solos/orphans are very widespread among Proteobacteria; however they are surprisingly understudied given that they are likely to play a major role in cell-cell communication in bacteria. Here we describe three simple methodologies/approaches that can be used in order to begin to study this subgroup of quorum sensing-related LuxR receptors.

Measurement of Fructose-Asparagine Concentrations in Human and Animal Foods.

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as its sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the nonenzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules that contribute to the color, taste, and aroma of heated foods. Among these end derivatives is acrylamide, which is present in some foods, especially in fried potatoes. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 400 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have F-Asn at this level or higher (fresh apricots, lettuce, asparagus, and canned peaches). Much higher concentrations (11 000-35 000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report reveals possible origins of F-Asn as a nutrient source for Salmonella and identifies foods that could be used together with a FraB inhibitor as a therapeutic agent for Salmonella.

Salmonella FraE, an Asparaginase Homolog, Contributes to Fructose-Asparagine but Not Asparagine Utilization.

Salmonella enterica can utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. This capability has been attributed to five genes in the fra locus. Previously, we determined that mutations in fraB (deglycase), fraD (kinase), or fraA (transporter) eliminated the ability of Salmonella to grow on F-Asn, while a mutation in fraE allowed partial growth. We hypothesized that FraE, a putative periplasmic fructose-asparaginase, converts F-Asn to NH4 + and fructose-aspartate (F-Asp). FraA could then transport F-Asp into the cytoplasm for subsequent catabolism. Here, we report that growth of the fraE mutant on F-Asn is caused by a partially redundant activity provided by AnsB, a periplasmic asparaginase. Indeed, a fraE ansB double mutant is unable to grow on F-Asn. Moreover, biochemical assays using periplasmic extracts of mutants that express only FraE or AnsB confirmed that each of these enzymes converts F-Asn to F-Asp and NH4 + However, FraE does not contribute to growth on asparagine. We tested and confirmed the hypothesis that a fraE ansB mutant can grow on F-Asp, while mutants lacking fraA, fraD, or fraB cannot. This finding provides strong evidence that FraA transports F-Asp but not F-Asn from the periplasm to the cytoplasm. Previously, we determined that F-Asn is toxic to a fraB mutant due to the accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp). Here, we found that, as expected, a fraB mutant is also inhibited by F-Asp. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella IMPORTANCE Salmonella is able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that the disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice and recognized this pathway as a novel and specific drug target. Here, we characterize the first step in the pathway wherein FraE hydrolyzes F-Asn to release NH4 + and F-Asp in the periplasm of the cell. A fraE mutant continues to grow slowly on F-Asn due to asparaginase activity encoded by ansB.

Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome.

BACKGROUND: Salmonella is one of the most significant food-borne pathogens to affect humans and agriculture. While it is well documented that Salmonella infection triggers host inflammation, the impacts on the gut environment are largely unknown. A CBA/J mouse model was used to evaluate intestinal responses to Salmonella-induced inflammation. In parallel, we evaluated chemically induced inflammation by dextran sodium sulfate (DSS) and a non-inflammation control. We profiled gut microbial diversity by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from fecal and cecal samples. These data were correlated to the inflammation marker lipocalin-2 and short-chain fatty acid concentrations. RESULTS: We demonstrated that inflammation, chemically or biologically induced, restructures the chemical and microbial environment of the gut over a 16-day period. We observed that the ten mice within the Salmonella treatment group had a variable Salmonella relative abundance, with three high responding mice dominated by >46% Salmonella at later time points and the remaining seven mice denoted as low responders. These low- and high-responding Salmonella groups, along with the chemical DSS treatment, established an inflammation gradient with chemical and low levels of Salmonella having at least 3 log-fold lower lipocalin-2 concentration than the high-responding Salmonella mice. Total short-chain fatty acid and individual butyrate concentrations each negatively correlated with inflammation levels. Microbial communities were also structured along this inflammation gradient. Low levels of inflammation, regardless of chemical or biological induction, enriched for Akkermansia spp. in the Verrucomicrobiaceae and members of the Bacteroidetes family S24-7. Relative to the control or low inflammation groups, high levels of Salmonella drastically decreased the overall microbial diversity, specifically driven by the reduction of Alistipes and Lachnospiraceae in the Bacteroidetes and Firmicutes phyla, respectively. Conversely, members of the Enterobacteriaceae and Lactobacillus were positively correlated to high levels of Salmonella-induced inflammation. CONCLUSIONS: Our results show that enteropathogenic infection and intestinal inflammation are interrelated factors modulating gut homeostasis. These findings may prove informative with regard to prophylactic or therapeutic strategies to prevent disruption of microbial communities, or promote their restoration.

The commensal microbiota exacerbate infectious colitis in stressor-exposed mice.

Exposure to a prolonged restraint stressor disrupts the colonic microbiota community composition, and is associated with an elevated inflammatory response to colonic pathogen challenge. Since the stability of the microbiota has been implicated in the development and modulation of mucosal immune responses, we hypothesized that the disruptive effect of the stressor upon the microbiota composition directly contributed to the stressor-induced exacerbation of pathogen-induced colitis. In order to establish a causative role for stressor-induced changes in the microbiota, conventional mice were exposed to prolonged restraint to change the microbiota. Germfree mice were then colonized by microbiota from either stressor-exposed or non-stressed control mice. One day after colonization, mice were infected with the colonic pathogen, Citrobacter rodentium. At six days post-infection, mice that received microbiota from stressor-exposed animals had significant increases in colonic pathology and pro-inflammatory cytokine (e.g. IL-1ß) and chemokine (e.g. CCL2) levels after C. rodentium infection in comparison with mice that received microbiota from non-stressed mice. 16S rRNA gene sequencing revealed that microbial communities from stressed mice did not have any detectable Bifidobacterium present, a stark contrast with the microbial communities from non-stressed mice, suggesting that stressor-induced alterations in commensal, immunomodulatory Bifidobacterium levels may predispose to an increased inflammatory response to pathogen challenge. This study demonstrates that the commensal microbiota directly contribute to excessive inflammatory responses to C. rodentium during stressor exposure, and may help to explain why gastrointestinal disorders are worsened during stressful experiences.

A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant.

Insertions in the Salmonella enterica fra locus, which encodes the fructose-asparagine (F-Asn) utilization pathway, are highly attenuated in mouse models of inflammation (>1000-fold competitive index). Here, we report that F-Asn is bacteriostatic to a fraB mutant (IC50 19 µM), but not to the wild-type or a fra island deletion mutant. We hypothesized that the presence of FraD kinase and absence of FraB deglycase causes build-up of a toxic metabolite: 6-phosphofructose-aspartate (6-P-F-Asp). We used biochemical assays to assess FraB and FraD activities, and mass spectrometry to confirm that the fraB mutant accumulates 6-P-F-Asp. These results, together with our finding that mutants lacking fraD or the fra island are not attenuated in mice, suggest that the extreme attenuation of a fraB mutant stems from 6-P-F-Asp toxicity. Salmonella FraB is therefore an excellent drug target, a prospect strengthened by the absence of the fra locus in most of the gut microbiota.