AcrAB efflux pump impacts on the survival of adherent-invasive Escherichia coli strain LF82 inside macrophages.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae. It consists of the AcrB transporter, which is embedded in the inner membrane, the AcrA adapter located in the periplasm, and the channel protein TolC responsible for the transport of substrates towards the extracellular environment. Besides conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here we report that the AcrAB pump heavily affects the infection process of the LF82 strain, the prototype of Adherent-Invasive Escherichia coli (AIEC) which are highly abundant in the ileal mucosa of Chron disease patients. We found that the deletion of genes encoding AcrA and/or AcrB leads to decreased survival of LF82 within macrophages. Ectopic AcrAB expression in a acrAB defective mutant restores the wild type condition. Furthermore, we demonstrate that inhibition of AcrB and replacement of the transporter with an unfunctional AcrB also interfere with bacterial viability inside macrophages. Overall, these data suggest a pivotal role of the AcrAB efflux pump in bacteria-host cell interactions also in AIEC.

Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae.

Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (µLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell.IMPORTANCE We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors.