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

Abstract

Staphylococcus aureus bi-component pore-forming toxins consist of S- and F-components, and form hetero-octameric beta-barrel pores on target blood cell membranes. Among them, γ-haemolysin (Hlg2 and F-component of Luk (LukF)) and LukED (LukE and LukD) possess haemolytic activity, whereas the Panton-Valentine leukocidin (LukS-PV and LukF-PV) 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 Hlg2 and LukE with that of LukS-PV abolished their haemolytic activity. Furthermore, LukS-PV gained haemolytic activity by Loop-4 exchange with Hlg2 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 Hlg2 and LukE recognize Duffy antigen receptor for chemokines on human erythrocytes, the ability of Loop-4 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.

Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes

“…S1A). Loop 1 of LukE contributed to hemolysis, but more minimally than the larger DR1 (16). Of note, Peng et.…”

“…As LukD and HlgB are critical for targeting erythrocytes (16,20,22), and S and F subunits can form active pores in non-canonical pairs (29,30), we next combined the gain of function chimeras with LukD and HlgB. Remarkably, we found that LukS E-DR1 exhibited full hemolytic activity when combined with LukD or HlgB, indistinguishable from WT LukED (Fig.…”

“…While conducting the experiments described herein, Peng and colleagues published a report also studying the sequence determinants of LukE involved in hemolysis (16). They used a similar approach, making LukE-LukS-PV chimeras, but swapped smaller loops at the tip of the rim domain.…”

“…LukE targets CXCR1 and 2 using residues 182-196 (8), and CCR5 using residues 57-75 (13). Interestingly, multiple regions of the HlgA rim and cap domains are involved in targeting erythrocytes through DARC (14)(15)(16).…”

Cytotoxicity analysis of staphylococcal bi-component β-pore forming toxins using the CHO cells expressing human lymphocyte receptor CCR5

“…Conversely, the loop structures in the rim domain of S-components have been investigated as candidate factor(s) that interact with target cell, because S-components lack the PC-binding site. Four loop structures have different amino acid sequences in the rim domain of Hlg2 and LukE, and these loops are involved in erythrocyte binding (Figure 1(a) and Figure S1) [8]. Hlg2 and LukE have been suggested to recognize the same receptor on the human erythrocytes via a different site of Duffy Antigen Receptor for Chemokines (DARC) [9].…”