Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

Abstract

Epsilon-toxin (ETX) is produced by types B and D strains of Clostridium perfringens, which cause fatal enterotoxaemia in sheep, goats and cattle. Previous studies showed that only a restricted number of cell lines are sensitive to ETX and ETX-induced hemolysis has not previously been reported. In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. Moreover, the mechanism of ETX-induced hemolysis was further explored. Recent studies showed that some bacterial toxins induce hemolysis through purinergic receptor (P2) activation. Hence, the function of purinergic receptors in ETX-induced hemolysis was tested, and we found that the non-selective P2 receptor antagonists PPADS inhibited ETX-induced lysis of human erythrocytes in a concentration-dependent manner, indicating that ETX-induced hemolysis requires activation of purinergic receptors. P2 receptors comprise seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14) receptor subtypes. The pattern of responsiveness to more selective P2-antagonists implies that both P2Y13 and P2X7 receptors are involved in ETX-induced hemolysis in human species. Furthermore, we demonstrated that extracellular ATP is likely not involved in ETX-induced hemolysis and the activation of P2 receptors. These findings clarified the mechanism of ETX-induced hemolysis and provided new insight into the activities and ETX mode of action.

Screening and purification of NanB sialidase from Pasteurella multocida with activity in hydrolyzing sialic acid Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal

“…As a lysis control, lysis buffer (Geneaid) was used. Toxicity was calculated using the following formula: 100 × (OD of sample—OD of negative control/ OD of lysis control—OD of negative control) 38 .…”

The MAL Protein, an Integral Component of Specialized Membranes, in Normal Cells and Cancer

“…Large-scale hemolysis occurs, sometimes producing severe anemia, in patients infected with C. perfringens [ 160 , 161 , 162 ]. ETX causes hemolysis in humans but not in other mammals [ 163 ], suggesting that ETX-binding receptors are present on human red blood cells but absent from those of other species. Consistent with these findings, a shorter 11-kDa MAL isoform ( Figure S2 ) was found in human erythrocytes but not in those of mouse or rat [ 164 ].…”

Screening and Purification of NanB Sialidase from Pasteurella Multocida with Activity in Hydrolyzing Sialic Acid Neu5Acα(2-6)Gal

“…As a lysis control, lysis buffer (Geneaid) was used. Toxicity was calculated using the following formula: 100 x (OD of sample -OD of negative control/ OD of lysis control -OD of negative control) (Gao et al 2018).…”