Residues 1 to 563 of α-hemolysin alone form a fully functional pore and their proper membrane insertion depends on the acylation status of the molecule

ABSTRACT: The acylated pore-forming Repeats in ToXin (RTX) cytolysins α-hemolysin (HlyA) and adenylate cyclase toxin (CyaA) bind primarily to β2 integrins of leukocytes. HlyA binds the common CD18 subunit of the β2 integrins and CyaA selectively binds the CD11b subunit of complement receptor 3 (CR3). However, both toxins can also bind and permeabilize membranes of a variety of nonmyeloid cells. We constructed HlyA1-563/CyaA860-1706 hybrid molecules activated by the CyaA-activating acyltransferase CyaC, or by the HlyA-activating acyltransferase HlyC. We show that the C-terminal portion of the HlyA molecule, comprising the acylated segment and the RTX domain (residues 564 to 1024), can be functionally swapped with the CyaC-activated acylated segment bearing palmitoylated Lys983 and the much larger RTX domain of CyaA. Compared to the CD18-interacting HlyA, the CR3-interacting HlyA1-563/CyaA860-1706 hybrid exhibited a selectively reduced cytotoxicity on human THP-1 monocytes and Chinese hamster ovary (CHO) cells expressing CR3 (CHO-CR3) or lymphocyte function-associated antigen 1 (CHO-LFA1). However, the mono-palmitoylated HlyA1-563/CyaA860-1706 hybrid remained fully hemolytic and cytolytic toward erythrocytes and mock-transfected CHO cells and exhibited a comparable membrane activity on artificial planar lipid membranes as the bi-myristoylated HlyA. Thus, regardless of the length or number of the attached fatty acyl chains, the CyaC-activated HlyA1-563/CyaA860-1706 hybrid and the intact HlyA were comparably active on cells or artificial membranes lacking β2 integrins. These data suggest that once the hydrophobic pore-forming domain comprising N-terminal half of HlyA is brought into close contact with the cell membrane by the mono-palmitoylated acylated segment of CyaA, it can efficiently form fully cytolytic HlyA-like membrane pores.

INSTRUMENT(S): Bruker Daltonics solarix series

ORGANISM(S): Escherichia Coli

TISSUE(S): Erythrocyte

DISEASE(S): Bordetella Parapertussis Whooping Cough

SUBMITTER: David Jurnečka  

LAB HEAD: Laboratory of Molecular Biology of Bacterial Pathogens

PROVIDER: PXD046160 | Pride | 2024-03-25

REPOSITORIES: Pride