Dynamic regulation of extracellular ATP in Escherichia coli

ALVAREZ CORA; CORRADI G; LAURI N.; MARGINEDAS-FREIXA I; LEAL DENIS MF; ENRIQUE NJ; MATE SM; MILESI V; OSTUNI MA; HERLAX V; SCHWARZBAUM P

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2017 vol. 474

0264-6021

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli , and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real time luminometry was used to measure ATPe kinetics, ATP release and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, coincubated with Caco-2 cells or in rat jejunum segments. In E. coli , addition of [γ-32P]ATP led to uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused acute 3-fold (MST7) and 7-fold (MEL) increase of [ATPe]. In OMVs ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. Addition of bacterial suspensions led to an 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release of Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.