S. aureus Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Abstract

Therapeutics for the treatment of cystic fibrosis have recently progressed to include drugs that directly increase CFTR channel activity; however, the influence of bacterial virulence factors on the efficacy of these potentiators is not well understood. One secreted protein of particular interest is sphingomyelinase (SMase), a sphingomyelin-specific phosphodiesterase that generates the signaling lipid ceramide and has been shown previously to inhibit CFTR channel activity in Xenopus laevis oocytes and the immortalized Calu3 epithelial cell line. In this study we performed mechanistic experiments to better understand how bacterial sphingomyelinase impacts CFTR channel function and how that inhibition affects the efficacy of the clinically prescribed CFTR potentiator, Kalydeco (VX770). Our data shown that purified S. aureus SMase is a gating modifier of CFTR wherein channels are inhibited at the plasma membrane independent of the R-domain, the best understood regulatory domain of CFTR. We also found that inhibition occurs in channels that are isolated from the enzyme and does not occur in excised patches when SMase is backfilled into the pipette suggesting that inhibition occurs through a cytosolic signaling event. Consistent with the idea that SMase activity modulates CFTR gating at the membrane, we found that inhibition efficacy was dependent upon CFTR conformational state. Specifically, we found that mutations known to increase channel activity significantly decreased the rate of inhibition by SMase. Finally, we found evidence that inhibited channels cannot be rescued by applying VX770 suggesting that inhibited channels are effectively removed from the druggable pool. Taken together, our data suggest that S. aureus SMase is a gating modifier of CFTR that inhibits channel activity in a manner that renders VX770 ineffective.