Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the Glycosylphosphatidylinositol-Binding Toxin Aerolysin

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59+ cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Paroxysmal Nocturnal Hemoglobinuria is rare cause for thrombosis of the intra‐abdominal veins in the ethnic Indian population – results fromFLAER‐based flowcytometry screening

“…Aerolysin, which is a toxin secreted by the bacterial pathogen Aeromonas hydrophila, is capable of killing target cells by forming channels in their membranes after binding to GPI‐anchored receptors, was originally shown by Brodsky et al . . PNH cells that lack the GPI‐anchored proteins are therefore resistant to the toxin.…”

Natural history of paroxysmal nocturnal hemoglobinuria clones in patients presenting as aplastic anemia

“…However, the natural history of PNH clones in patients with AA following HiCy therapy has not been documented. We developed a highly sensitive and specific assay to detect PNH blood cells based on the properties of the bacterial toxin proaerolysin (19, 20). Since 1998, we have used this assay to detect small PNH clones in patients with AA (13).…”

Generation of Glycosylphosphatidylinositol Anchor Protein-Deficient Blood Cells From Human Induced Pluripotent Stem Cells

“…We next used FACS sorting to collect RFP + /CD59 − and RFP + /CD59 + undifferentiated FPHR‐KP cells, cultured them in differentiation‐inducing medium without or with Dox for up to 14 days, and counted the percentage of EBs with blood cells in 96‐well plates (Fig. 5A, 5B). The RFP + /CD59 − without Dox formed abnormal EBs and did not generate blood cells.…”

“…A hallmark of PNH blood cells is that they are resistant to the bacterial channel‐forming toxin proaerolysin [5, 29, 37]. Proaerolysin uses GPI‐AP as its receptor.…”

“…GPI‐anchored proteins (GPI‐APs) function as complement regulatory proteins, enzymes, blood group antigens, receptors, and adhesion molecules. GPI‐APs also serve as receptors for proaerolysin, a pore‐forming bacterial toxin secreted by Aeromonas hydrophila [4, 5]. The GPI anchor is synthesized in the endoplasmic reticulum membrane and involves at least 10 reactions and more than 20 different gene products [3].…”