The broadly conserved regulator PhoP links pathogen virulence and membrane potential in Escherichia coli

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87159/1/j.1365-2958.2011.07804.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/87159/2/MMI_7804_sm_FigS1-4-TabS1.pd

Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection

Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-γ and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections

An intron variant of the GLI family zinc finger 3 (GLI3) gene differentiates resistance training-induced muscle fiber hypertrophy in younger men

We examined the association between genotype and resistance training-induced changes (12 wk) in dual x-ray energy absorptiometry (DXA)-derived lean soft tissue mass (LSTM) as well as muscle fiber cross-sectional area (fCSA; vastus lateralis; n = 109; age = 22 ± 2 y, BMI = 24.7 ± 3.1 kg/m2). Over 315 000 genetic polymorphisms were interrogated from muscle using DNA microarrays. First, a targeted investigation was performed where single nucleotide polymorphisms (SNP) identified from a systematic literature review were related to changes in LSTM and fCSA. Next, genome-wide association (GWA) studies were performed to reveal associations between novel SNP targets with pre- to post-training change scores in mean fCSA and LSTM. Our targeted investigation revealed no genotype-by-time interactions for 12 common polymorphisms regarding the change in mean fCSA or change in LSTM. Our first GWA study indicated no SNP were associated with the change in LSTM. However, the second GWA study indicated two SNP exceeded the significance level with the change in mean fCSA (P = 6.9 × 10–7 for rs4675569, 1.7 × 10–6 for rs10263647). While the former target is not annotated (chr2:205936846 (GRCh38.p12)), the latter target (chr7:41971865 (GRCh38.p12)) is an intron variant of the GLI Family Zinc Finger 3 (GLI3) gene. Follow-up analyses indicated fCSA increases were greater in the T/C and C/C GLI3 genotypes than the T/T GLI3 genotype (P \u3c.05). Data from the Auburn cohort also revealed participants with the T/C and C/C genotypes exhibited increases in satellite cell number with training (P \u3c.05), whereas T/T participants did not. Additionally, those with the T/C and C/C genotypes achieved myonuclear addition in response to training (P \u3c.05), whereas the T/T participants did not. In summary, this is the first GWA study to examine how polymorphisms associate with the change in hypertrophy measures following resistance training. Future studies are needed to determine if the GLI3 variant differentiates hypertrophic responses to resistance training given the potential link between this gene and satellite cell physiology

Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Arginine promotes Proteus mirabilis motility and fitness by contributing to conservation of the proton gradient and proton motive force

Swarming contributes to Proteus mirabilis pathogenicity by facilitating access to the catheterized urinary tract. We previously demonstrated that 0.1–20 mmol/L arginine promotes swarming on normally nonpermissive media and that putrescine biosynthesis is required for arginine‐induced swarming. We also previously determined that arginine‐induced swarming is pH dependent, indicating that the external proton concentration is critical for arginine‐dependent effects on swarming. In this study, we utilized survival at pH 5 and motility as surrogates for measuring changes in the proton gradient (ΔpH) and proton motive force ( μ H + ) in response to arginine. We determined that arginine primarily contributes to ΔpH (and therefore μ H + ) through the action of arginine decarboxylase ( speA ), independent of the role of this enzyme in putrescine biosynthesis. In addition to being required for motility, speA also contributed to fitness during infection. In conclusion, consumption of intracellular protons via arginine decarboxylase is one mechanism used by P. mirabilis to conserve ΔpH and μ H + for motility. We previously determined that Proteus mirabilis swarming can be initiated under normally nonpermissive conditions in response to cues, such as L‐arginine, and this process requires putrescine biosynthesis or exogenously supplied putrescine. In this study, we describe a mechanism by which P. mirabilis utilizes L‐arginine and arginine decarboxylase (SpeA) for conservation of proton motive force, affecting both motility and fitness independent of the role of this enzyme in putrescine biosynthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109334/1/mbo3194.pd

Fluorescence difference in gel electrophoresis (2D-DIGE) of UPEC cytoplasmic proteins during growth in urine.

<p>Soluble proteins (50 µg) from <i>E. coli</i> CFT073 cultured in urine were labeled with Cy3 (green), from CFT073 grown in LB with Cy5 (red), and the pooled internal standard representing an equal amount of urine and LB soluble proteins with Cy2 (blue). The labeled proteins (150 µg) were pooled and applied to a pH 4–7 IPG strip and second dimension 10% SDS-PAGE. Green spots indicate protein features induced in urine; red spots represent proteins induced in LB medium.</p

Growth of central metabolism mutants <i>in vitro</i> and <i>in vivo</i>.

<p>Growth of central metabolism mutants <i>in vitro</i> and <i>in vivo</i>.</p

<i>In vivo</i> fitness for select 2D-DIGE mutants.

a<p>Competitive Index, determined by dividing the ratio of wild-type to mutant at 48 hpi by the ratio present in the inoculum. Significant CI>1 indicates mutant has a fitness defect.</p>b<p><i>P</i>-values determined by Wilcoxon matched pairs test. Significant <i>P</i>-values are bolded.</p

<i>In vivo</i> complementation of UPEC Δ<i>pckA</i>.

<p>Individual female mice were transurethrally inoculated with 2×10⁸ CFU of a 1∶1 mixture of wild-type CFT073 containing pGEN empty vector and Δ<i>pckA</i> containing pGEN empty vector or pGEN-<i>pckA</i>. At 48 hpi, bladders were aseptically removed, homogenized, and plated on LB with ampicillin or LB containing ampicillin and kanamycin to determine viable counts of wild-type (closed symbols) and mutant strains (open symbols), respectively. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (<i>P</i><0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.</p