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 α_L, α_M, or α_X 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.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 β₂ 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.

Keywords: RTX toxin; cytotoxicity; hemolysin; integrins; leukotoxin.