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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0264-8) contains supplementary material, which is available to authorized users.

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.

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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 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 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 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, , and can utilize F-Asn, but cannot; and some subspecies can utilize F-Asn; and some and strains can also utilize F-Asn. Within, the host-adapted serovars Typhi and Paratyphi A have lost the ability to utilize F-Asn. Fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and it is also a nutrient source for , a foodborne pathogen. Here, we determined that among the normal intestinal microbiota, there are species of that encode the enzymes required for F-Asn utilization. Using complementary experimental approaches, we have confirmed that three members of , two members of, and two members of can indeed utilize F-Asn. The spp. likely compete with 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 species. This study identifies the potential off-target organisms that need to be considered when developing therapeutics directed at FraB.

elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to during infection is dependent upon 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 encode F-Asn utilization pathways and that they are eliminated from highly inflamed-infected mice. Collectively, our studies identify the source of F-Asn as the diet and that -mediated inflammation is required to eliminate competitors and allow the pathogen nearly exclusive access to this nutrient.

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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 (11000–35000 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.

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