Salmonella Typhimurium is a pathogenic bacterium capable of colonizing the mammalian gastrointestinal tract, causing salmonellosis, a diarrheal disease. Schubert et al, aimed to investigate which nutrients S. Typhimurium utilizes during colonization. To systematically analyze different nutrient sources, we generated a library of 35 S. Typhimurium mutants, each deficient in the ability to metabolize a specific nutrient. To track each mutant, we incorporated a neutral genetic tag into the genome of S. Typhimurium, which does not affect bacterial fitness but allows for the precise identification and quantification of individual strains during experiments. This approach enabled us to monitor how different nutrient utilization pathways contribute to S. Typhimurium colonization.

Our lab has extensive experience in S. Typhimurium screening in mouse models, particularly using randomly barcoded transposon mutant libraries (Nguyen et al, 2024). In these libraries, genes are randomly inactivated with a unique tag, generating mutant pools containing thousands of different S. Typhimurium mutants. However, this approach presents several challenges, including bioinformatics complexity, as the barcode must first be mapped to its corresponding inactivated gene, adding computational workload. Additionally, since gene disruptions are random, key genes or pathways of interest may be missing. This method also requires more mice and deeper sequencing to generate sufficient data for assessing mutant fitness, making it costly. To address these limitations, we took a targeted approach. Instead of random mutagenesis, we specifically constructed S. Typhimurium mutants deficient in nutrient utilization. Each mutant was then tagged with a neutral genetic marker, the WISH-tag, allowing precise tracking of strain abundance. This method, previously established and published in Nature Microbiology, ensures a controlled, cost-effective, and biologically relevant strategy to study nutrient-driven fitness in the gut environment. This approach is undoubtedly more labor-intensive and requires the ability to genetically manipulate your model organism. However, if these conditions are met, it offers a highly controlled, cost-effective, and biologically relevant strategy for studying microbial fitness and nutrient utilization. In our lab, most members have now utilized this S. Typhimurium mutant pool in various mouse models, including competition experiments with other bacteria and studies in immunodeficient mice to assess how the host influences S. Typhimurium mutant fitness. A key advantage of this approach is its scalability—new WISH-barcoded S . Typhimurium mutants can be seamlessly integrated, allowing us to expand the scope of investigations and address a broader range of scientific questions.

In our current study, we utilized established mouse models with varying levels of colonization resistance—the ability of the gut microbiota to prevent S. Typhimurium infection. In models with high colonization resistance, S. Typhimurium cannot reliably establish itself in the gut. Thus, we selected mouse models with either a completely absent microbiota (germ-free mice), an antibiotic-disrupted microbiome, or a simplified microbiota composed of only a few bacterial species. We prepared the S. Typhimurium mutant pool and infected our mouse models to study nutrient utilization during colonization. At specific time points post-infection, we collected fecal samples, extracted bacterial DNA, and quantified the abundance of each unique barcode. Since the pool also contained wild-type S. Typhimurium, we could directly compare how mutants deficient in utilizing specific nutrients colonized the gut relative to the wild type.

We identified D-glucose, D-mannose, and D-fructose as key nutrient sources for S. Typhimurium. Notably, D-glucose and D-fructose are also primary energy sources for humans and are abundant in the diet. However, for some time, it was unclear whether free monosaccharides were readily available in the gastrointestinal tract, particularly in the cecum of mice, which serves as the primary site of S. Typhimurium infection, as opposed to the colon in humans. Enterobacteriaceae, including S. Typhimurium, are highly adapted to D-glucose metabolism. However, the ecological niche where D-glucose is sufficiently abundant to drive this adaptation remained unclear. Recent studies, including work from the Bäumler lab, have confirmed that D-glucose and other monosaccharides are present at sufficient concentrations in the gut to support S. Typhimurium growth, explaining the pathogen’s metabolic adaptation.

In Schubert et al., we demonstrated that the ability to utilize D-glucose, D-mannose, and D-fructose is widely conserved across different genera of Enterobacteriaceae, including Escherichia and Shigella. In a separate study, we broadened our analysis to investigate carbohydrate utilization systems across 16 genera within the Enterobacteriaceae family. Our findings confirmed that the ability to utilize D-glucose, D-mannose, D-galactose, and D-fructose is highly conserved across Enterobacteriaceae. Understanding which nutrients bacteria, particularly pathogenic bacteria, consume in the gut is crucial. This knowledge provides a foundation for developing potential therapeutic strategies aimed at either preventing S. Typhimurium colonization or restricting its expansion within the gut environment.

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