Effects of cations and osmotic protectants on cytolytic activity of Actinobacillus actinomycetemcomitans leukotoxin.
Actinobacillus actinomycetemcomitans leukotoxin permeabilized the plasma membrane of HL-60 promyelocytic leukemia cells, resulting in colloid osmotic lysis. These events were associated with efflux of 51chromium (from prelabeled cells), influx of propidium iodide, and ultrastructural evidence of cellular damage. Target cell lysis was inhibited by procedures which may interfere with the initial interaction of the toxin with the plasma membrane. For example, washing cultures (to dilute and remove toxin) or the addition of monoclonal antibodies (to neutralize toxin) or trypsin (to inactivate toxin) limited lysis when undertaken within the first 5 min of the reaction. The extent of injury was also diminished when radiolabeled HL-60 cells were exposed to toxin in the presence of unlabeled, toxin-sensitive cells (e.g., HL-60 cells or human neutrophils) or certain toxin-resistant target cells (e.g., human K562 erythroleukemia cells). This suggests that the association of the toxin with the cell membrane may not be sufficient to cause lysis without activation of additional effector mechanisms. The addition of specific trivalent (e.g., La3+) or divalent (e.g., Ca2+ and Zn2+) cations to toxin-treated cells appeared to enhance their capacity to repair or minimize the extent of toxin-mediated membrane damage. Depending on size, certain saccharides served as osmotic protectants: maltose almost completely inhibited radiolabel release, while smaller molecules provided correspondingly less protection. The results imply that the leukotoxin has membranolytic activity, producing pores in target cells with a functional diameter approximately the size of maltose (0.96 nm).
Iwase
M
M
Department of Oral Surgery, School of Dental Medicine, Showa University, Tokyo, Japan.
Lally
E T
ET
Berthold
P
P
Korchak
H M
HM
Taichman
N S
NS
D000188:Actinobacillus / Q000472:pathogenicity*; D000818:Animals; D000911:Antibodies, Monoclonal; D002412:Cations / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005098:Exotoxins / Q000037:antagonists & inhibitors*; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000187:drug effects*; D008854:Microscopy, Electron; D009005:Monosaccharides / Q000494:pharmacology; D009994:Osmolar Concentration; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002412:Cations; D005098:Exotoxins; D009005:Monosaccharides; C018579:leukotoxin; D014357:Trypsin