Rim domain loops of Staphylococcal β-pore forming bi-component toxin S-components recognize target human erythrocytes in a coordinated manner.

Staphylococcus aureus hi-component pore-forming toxins consist of S- and F-components, and form hetero-octameric beta-barrel pores on target blood cell membranes. Among them, gamma-haemolysin (bHlg2 and F-component of Luk (LukF)) and LukED (LukE and LukD) possess haemolytic activity, whereas the Panton-Valentine leukocidin (LukS-PV and LukFPV) does not lyse human erythrocytes. Here, we focussed on four loop structures in the rim domain of S-component, namely loops -1, -2, -3 and -4, and found that replacement of Loop-4 in both bHlg2 and LukE with that of LukS-PV abolished their haemolytic activity. Furthermore, LukS-PV gained haemolytic activity by Loop-4 exchange with H1g2 or LukE, suggesting that Loop-4 of these S-components determined erythrocyte specificity. LOOP-1 and -2 enhanced the erythrocytes-binding ability of both components. Although bHlg2 and LukE recognize Duffy antigen receptor for chemokines on human erythrocytes, the ability of Loop4 was not complementary between Hlg2 and LukE. Exchange of Hlg2 with LukE Loop-4 showed weaker activity than intact Hlg2, and LukE mutant with Hlg2 Loop-4 lost its haemolytic activity in combination of LukD. Interestingly, the haemolytic activities of these Loop-4 exchange mutants were affected by F-component, namely LukF enhanced haemolytic activities of these Hlg2 and LukE Loop-4 mutants, and also haemolytic activity of LukS-PV mutant with LukE Loop-4.