The Extracellular Domain Of The Beta(2) Integrin Beta Subunit (Cd18) Is Sufficient For Escherichia Coli Hemolysin And Aggregatibacter Actinomycetemcomitans Leukotoxin Cytotoxic Activity

The Escherichia coli hemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HIyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA from Aggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the beta(2) integrin beta subunit. Delta beta(2) cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HIyA at high concentrations. Similarly, Delta beta(2) cells are more resistant than wild-type U-937 cells to LtxA, as Delta beta(2) cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single beta(2) integrin alpha subunit, or even all four alpha subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the beta(2) subunit, but not to alpha(L), alpha(M), or alpha(X) in far-Western blots. Genetic complementation of Delta beta(2) cells with either beta(2) or beta(2)with a cytoplasmic tail deletion restores HIyA and LtxA sensitivity, suggesting that beta(2) integrin signaling is not required for cytotoxicity. Finally, beta(2 )mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Delta beta(2) cells are equally sensitive to Staphylococcus aureus alpha-toxin and Proteus mirabilis HpmA. Our studies show two RTX toxins use the beta(2) integrin beta subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCE Urinary tract infections are one of the most common bacterial infections worldwide. Uropathogenic Escherichia coli strains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenic Escherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the beta 2 integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. The E. coli hemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.