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Microcolony-seq applied on a clinical urinary tract infection

ABSTRACT: Microcolony-seq applied on a clinical urinary tract infection

PROVIDER: PRJEB73604 | ENA |

REPOSITORIES: ENA

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Microcolony-seq applied on a clinical urinary tract infection

Project description:Urine was obtained from a patient with a urinary tract infection and frozen in 15% glycerol at -80C. For the Microcolony-seq experiment a sample from the frozen urine was serially diluted four times 10-fold and plated on an LB agar plates and incubated at 37C. After 8 hours of incubated microcolonies were visible. Twenty microcolonies were picked and subjected to the Microcolony-seq pipeline and four additional microcolonies were mixed and separated to four Eppendorf tubes and served as the technical replicates of the experiment. RNA was extracted with 1 mL TriReagent (Sigma-Aldrich) per sample. RNA quality was assessed by Nanodrop and by Bioanalyzer using the Agilent RNA 6000 Pico Kit (5067-1513). rRNA depletion was done by using the DIY rRNA depletion method, using the rRNA sequence probe set designed for E. coli K-12. RNA-seq libraries were constructed based on the RNAtag-Seq protocol with several modifications. The rRNA depletion step was done first following by the fragmentation step in the RNAtag-Seq protocol. Then the first ligation was carried out and the rest of the RNAtag-Seq protocol was followed. Libraries were single-end sequenced using the Nextseq500 Sequencer (Illumina).

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Obesity promotes urinary tract infection by disrupting urothelial immune defenses

Project description:Obesity promotes urinary tract infection by disrupting urothelial immune defenses

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Obesity promotes urinary tract infection by disrupting urothelial immune defenses

Project description:Obesity is a significant public health concern associated with increased infection risk, but the mechanisms remain unclear. Using a diet induced obesity mouse model, we investigate how obesity impacts urinary tract infection (UTI) susceptibility and bladder urothelial defenses. High fat diet-fed female and male C57BL/6 mice exhibit increased susceptibility to uropathogenic E. coli (UPEC) following experimental UTI. Transcriptomic analysis of bladder urothelial cells reveals sex-specific gene expression changes, but both sexes share activation of focal adhesion and extracellular matrix signaling. Western blot and immunostaining confirm activation of focal adhesion kinase (FAK), a central component of the focal adhesion pathway, in the bladders of obese female and male mice. Mechanistically, primary human urothelial cells overexpressing FAK exhibit increased UPEC invasion. These findings demonstrate that obesity enhances UTI susceptibility and identify FAK as a conserved pathway disrupted by obesity, contributing to increased UPEC vulnerability.

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