Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars 1, increases the duration for which patients shed the pathogen in their feces and may on occasion produce a bacteriologic and symptomatic relapse 2,3. These antibiotic-induced changes in the gut microbiota can be studied in mice, where the disruption of a balanced microbial community by treatment with streptomycin leads to an expansion of S. enterica serovars in the large bowel 4. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the cecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine cecum. S. Typhimurium utilized galactarate and glucarate within the gut lumen of streptomycin pre-treated mice and genetic ablation of the respective catabolic pathways reduced its competitiveness. Our results identify a host-mediated oxidation of carbohydrates in the gut as a novel mechanism for post-antibiotic pathogen expansion.
k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.
Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB. Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field. IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.
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Soy consumption is associated with thyroid disorders such as hypothyroidism, goiter, and autoimmune thyroid disease (ATD) as well as increased iodine requirement in certain cases. However, the anti-thyroid component(s) in soy are yet to be identified and the molecular mechanism(s) involved remain unclear. This study examined the effects of soy isoflavones (ISF) on iodide uptake and expression of thyroglobulin (Tg) and sodium/iodide symporter (NIS) in thyrocytes. Fischer rat thyroid cells (FRTL) were treated with Novasoy (a soy alcohol extract containing 30% ISF) or major ISF aglycones or glycosides for 24 h. Iodide uptake was measured by a colorimetric assay. The protein level of Tg and NIS was measured by Western blotting. Cytotoxicity of tested compounds was determined by the MTT cell proliferation assay. Iodide uptake in FRTL cells was dose-dependently suppressed by Novasoy added into the cell culture (10, 25, or 50 µg/mL, P < 0.05). However, neither the major ISF aglycones nor glycosides alone or in combination had similar effects. Novasoy (up to 200 µg/mL) had no cytotoxic effect. Novasoy (1, 10, and 50 µg/mL) and genistein (1 and 10 µM) markedly increased the protein content of a 40 kDa Tg fragment (P40, a known autoimmunogen) and non-glycosylated NIS in the FRTL cells (P < 0.05). Overall, this study demonstrated that the alcohol soluble component(s) other than the major ISF in soy remarkably inhibited iodide uptake in the FRTL cells. Soy ISF, particularly genistein, induced the production of P40, which might be responsible for the higher incidence of ATD reported in soy infant formula-fed children.
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