The extracellular domain of the β2 integrin β subunit (CD18) is sufficient for Escherichia coli hemolysin and Aggregatibacter actinomycetemcomitans leukotoxin cytotoxic activity

Abstract

The Escherichia coli hemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA 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 β2 integrin β subunit. Δβ2 cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2 cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2 cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2 integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2 subunit, but not to αL, αM, or αX in far-Western blots. Genetic complementation of Δβ2 cells with either β2 or β2 with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2 integrin signaling is not required for cytotoxicity. Finally, β2 mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2 cells are equally sensitive to Staphylococcus aureus α-toxin and Proteus mirabilis HpmA. Our studies show two RTX toxins use the β2 integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.