Human alpha-defensins inhibit hemolysis mediated by cholesterol-dependent cytolysins.
Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.
Lehrer
Robert I
RI
Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA. rlehrer@mednet.ucla.edu
Jung
Grace
G
Ruchala
Piotr
P
Wang
Wei
W
Micewicz
Ewa D
ED
Waring
Alan J
AJ
Gillespie
Eugene J
EJ
Bradley
Kenneth A
KA
Ratner
Adam J
AJ
Rest
Richard F
RF
Lu
Wuyuan
W
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D002784:Cholesterol / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006360:Heat-Shock Proteins / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000633:toxicity*; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000633:toxicity*; D008969:Molecular Sequence Data; D011817:Rabbits; D044967:Serum / Q000502:physiology; D013301:Streptolysins / Q000633:toxicity*; D023084:alpha-Defensins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008562:Membrane Glycoproteins; D013301:Streptolysins; D023084:alpha-Defensins; C498000:anthrolysin O, Bacillus anthracis; C032290:plY protein, Streptococcus pneumoniae; D002784:Cholesterol; C038705:hlyA protein, Listeria monocytogenes