IDENTIFICATION OF A BACTERIAL INHIBITOR OF PROTEIN-KINASES - MECHANISM AND ROLE IN HOST-CELL INVASION

We show that Escherichia coli produce a factor that inhibits the activ ity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immun oassay specific for tyrosine kinase activity and inhibition of the tyr osine kinase p56(lck), we purified this factor to apparent homogeneity . Analysis of trypsin-digested fragments by mass spectrometry identifi ed the inhibitor as the bacterial periplasmic protein UDP-sugar hydrol ase, an enzyme with potent and nonspecific 5'-nucleotidase activity. O verexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming t he identity of the inhibitor. The kinase inhibitory activity appears t o be due to the formation of adenosine, which we show is inhibitory fo r p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpr ession of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers a nd corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important fa ctor affecting host cell function following intracellular bacterial in fection.