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Antimicrobial Agents and Chemotherapy, May 2004, p. 1561-1569, Vol. 48, No. 5
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.5.1561-1569.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
In Vitro Antifungal Activities of Inhibitors of Phospholipases from the Fungal Pathogen Cryptococcus neoformans
Ranjini Ganendren, Fred Widmer, Vatsala Singhal, Christabel Wilson, Tania Sorrell, and Lesley Wright*Centre for Infectious Diseases and Microbiology, University of Sydney at Westmead, and Department of Infectious Diseases, Westmead Hospital, Westmead, NSW 2145, Australia
Received 26 September 2003/ Returned for modification 24 December 2003/ Accepted 20 January 2004
Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A2 at 0.25 µM. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology, Level 3, ICPMR Building, Westmead Hospital, Westmead, Sydney 2145, NSW, Australia. Phone: 612-98457367. Fax: 612-98915317. E-mail: lesleyw{at}icpmr.wsahs.nsw.gov.au.
Antimicrobial Agents and Chemotherapy, May 2004, p. 1561-1569, Vol. 48, No. 5
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.5.1561-1569.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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