Chemical inhibition of alpha-toxin, a key corneal virulence factor of Staphylococcus aureus.
OBJECTIVE
alpha-Toxin mediates extreme corneal damage during Staphylococcus aureus keratitis. Chemical inhibition of this toxin was sought to provide relief from toxin-mediated pathology.


METHODS
Inhibition of alpha-toxin by phosphate-buffered saline (PBS), 0.1% methyl-beta-cyclodextrin (CD), or CD plus cholesterol (0.1%, CD-cholesterol) was assayed by hemolysis of rabbit erythrocytes. Pathologic changes in rabbit corneas injected with 12 hemolytic units of alpha-toxin suspended in PBS, 1% CD, or 1% CD-cholesterol were compared over time. Rabbit corneas injected with 10(2) colony forming units (CFU) of S. aureus were treated from 7 to 13 hours postinfection (PI) with a total of 15 drops of CD-cholesterol, CD, or PBS. Slit lamp examination (SLE) and measurement of erosions were performed at 13 hours PI and bacteria were quantified at 14 hours PI.


RESULTS
Toxin-mediated lysis of erythrocytes was inhibited up to 16,000-fold in the presence of CD-cholesterol compared with CD or PBS. Eyes injected with alpha-toxin mixed with CD-cholesterol had, at 7 hours postinjection, significantly smaller erosions than eyes injected with alpha-toxin in PBS or alpha-toxin mixed with CD (P = 0.0090 and P = 0.0035, respectively). Eyes infected with S. aureus and treated with CD-cholesterol had significantly lower SLE scores than eyes treated with CD or PBS (P <or= 0.0103 and P <or= 0.0017, respectively); however, there were no differences in the number of bacteria present (P >or= 0.0648).


CONCLUSIONS
CD-cholesterol is a potent inhibitor of alpha-toxin activity in vitro and an effective means to arrest corneal damage during S. aureus keratitis.
McCormick
Clare C
CC
Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA. cmccormick@microbio.umsmed.edu
Caballero
Armando R
AR
Balzli
Charles L
CL
Tang
Aihua
A
O'Callaghan
Richard J
RJ
D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors*; D002784:Cholesterol / Q000494:pharmacology*; D003315:Cornea / Q000382:microbiology; D003320:Corneal Ulcer / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control*; D004195:Disease Models, Animal; D004359:Drug Therapy, Combination; D004912:Erythrocytes / Q000187:drug effects; D005098:Exotoxins / Q000037:antagonists & inhibitors; D015818:Eye Infections, Bacterial / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control*; D006460:Hemolysin Proteins / Q000037:antagonists & inhibitors*; D006461:Hemolysis; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology; D013203:Staphylococcal Infections / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control; D013208:Staphylococcal Toxoid / Q000037:antagonists & inhibitors*; D013211:Staphylococcus aureus / Q000502:physiology; D014774:Virulence / Q000502:physiology; D037521:Virulence Factors / Q000037:antagonists & inhibitors*; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D001427:Bacterial Toxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D013208:Staphylococcal Toxoid; D037521:Virulence Factors; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C021091:staphylococcal alpha-toxin; D012965:Sodium Chloride; D002784:Cholesterol