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Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
Changes in ppGpp levels impact gene expression and virulence features of Adherent-Invasive Escherichia coli strain
Llorenç Fernández-Coll1,2, Queralt Bonet-Rossinyol1, Mireia López-Siles1
1Microbiology of Intestinal Diseases, Biology Department, University of Girona, Girona, Spain.
Abstract:
Strain LF82 belongs to the Adherent-Invasive Escherichia coli (AIEC) pathotype, found in 21% to 63% of Crohn's disease (CD) patients. Many studies have explored its specific virulence mechanisms, but AIEC isolates are genomically similar to non-invasive isolates of E. coli, suggesting a differential gene expression. Here we study the impact of the global regulator ppGpp in the expression of AIEC's virulence factors and its potential for therapeutic and diagnostic purposes. Levels of ppGpp were determined from P-33 labelled cultures of LF82 and MG1655 (commensal strain) and differences in gene expression during infection of intestinal epithelial cells were assessed through RNAseq. Our study revealed that ppGpp is responsible for the expression of different genes involved in AIEC's pathogenicity (Type I fimbriae, T6SS or Vat), and other global regulators (as CRP or c-di-GMP). AIEC characteristics and virulence features (biofilm formation and motility) were determined, showing that ppGpp is essential for LF82 to adhere to and invade epithelial intestinal cells, as well as to survive in macrophages. Lastly, virulence was evaluated in vivo using the Galleria mellonella larvae model, showing a decrease in virulence in the absence of ppGpp. Analysis of a panel of differentially expressed genes across a collection of 7 AIEC strains and 6 non-AIEC strains, identified two candidate biomarkers which expression show a discriminatory potential for AIEC identification in the future. Altogether, our findings establish ppGpp as a key regulator of the AIEC virulence program and provide novel targets for both therapeutic intervention and diagnostic development.
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