Vibrio alginolyticus thermolabile hemolysin (TLH) induces apoptosis, membrane vesiculation and necrosis in sea bream erythrocytes

Abstract

Nucleated erythrocytes of silver sea bream (Sparus sarba) were found to enter a unique death mode exhibiting both features of apoptosis and necrosis after exposure to a recombinant thermolabile hemolysin (TLH) of Vibrio alginolyticus. TLH induced significant increases in caspase-3, -8, -9/6 activities followed by apoptotic DNA fragmentation (TUNEL and DNA ladder assays). Apart from these typical apoptotic events, membrane vesiculation consisting of protrusions on the plasma membrane was observed using transmission electron microscopy. Protrusions on plasma membrane became detached as particles which were confirmed to be plasma membrane fragments using an acetylcholinesterase (AChE) assay for quantifying membrane-associated AChE. This was followed by post-apoptotic necrosis as evidenced by disintegration of the plasma membrane leading to leakage of hemoglobin and other cytoplasmic contents. The nuclear envelope remained intact but nuclear contents became homogeneous due to chromatin fragmentation. These events resulted in marked changes in the distribution of cell size of the erythrocyte population as shown by flow cytometry. A correlation among membrane vesiculation, apoptosis and post-apoptotic necrosis appears to exist as the magnitudes of these events were elevated synchronously when the TLH dosage was increased.