Molecular Functionality of the Urophatogenic RTX Toxin Haemolysin A from E. coli

Haemolysin A (HlyA) is a 110 kDa toxin produced by certain pathogenic E. coli strains. HlyA is a member of the RTX (Repeats In Toxins) protein family and capable of lysing erythrocytes. The toxin is secreted by a Type I secretion system in one step through both lipid bilayers. Involved in this secretion mechanism are the ABC-type transporter HlyB, the membrane fusion protein HlyD and the outer membrane protein TolC, which form the ternary Hly secretion complex. The restricted diameter of the TolC outer membrane channel however indicates that HlyA is most likely exported in a (largely) unfolded state. Upon secretion, extracellular Ca²⁺ binds to the RTX domain thereby inducing the folding of HlyA to its biologically active conformation. In addition, HlyA toxin activity requires acylation of two specific lysine residues. Acylation is carried out in the cytoplasm by the acyltransferase HlyC in conjunction with the acyl-carrier protein (ACP). The molecular mechanisms by which HlyA is acylated, secreted and is folded to its native form are still largely unknown.

To study the acylation and folding of HlyA in vitro, proHlyA (un-acylated), mature HlyA, HlyC and ACP need to be purified. First experiments indicate that cells lacking HlyC produce and secrete proHlyA in high amounts. ProHlyA could be purified to near homogeneity with high yield (>60 mg/L). Initial folding experiments with urea indicate that Ca²⁺ levels drastically influence the stability of the secreted proHlyA.