The echinocandins (ECs) are a new antifungal group that includes caspofungin, micafungin, and anidulafungin. ECs represent a profoundly important development in antifungal chemotherapy because they are generally fungicidal, non-toxic, and remain active versus strains that acquire resistance to other antifungals. These advantages stem from the unique mechanism ofEC action involving inhibition of fungal p-l ,3-glucan synthase and hence cell wall synthesis. Reduced echinocandin susceptibility (RES) can develop in normally susceptible fungi such iIS Candida albicans by mutation of FKSl encoding the major glucan synthase, but fOrtw'l ately such mutations occur only rarely. However, a number ofless common but often more lethal fungal pathogens demonstrate intrinsic RES of varying levels. FusarIUm, Scedosporium, Cryptococcus, Trichosporon, and zygomycete s~ies are characterized by high-level intrinsic RES, while Aspergillus specics and Candida parapsilosis exhibit low-level intrinsic RES. Intrinsic RES represents a serious limitation to EC use, since antifungal treatment is often empirical and antifungaJ prophylaxis is increasingly relied upon in high risk patients. To address this limitation, we must understand the basis for intrinsic RES. Our central hypothesis is that intrinsic RES, as in mutationally acquired RES, is mediated by Fks protein sequences. Support for this hypothe;is has been provided by Our recently published studies examining intrinsic resistance ofC. paraosilosis (GarciaEffron et al .. 200S. Me 52:2305) and Fusariwn solani (Katiyar and &i1ind. 2009. MC in press). We propose to fyrfug tcst this by using the yeast Saccharomyces cerevisiae as model and surrogate host, through the following Aims: (I) Mutatiorlally defIne the EC target in S. cerevisiae FksJ. Begirlning with our recently expanded database of spontaneous RES mutations (manuscripts in preparation).
this Aim will rely on a new method for peR-based site-direcled mutagenesis (pSM) that is efficient, versatile... and immune to ex""""""""l)ression artifacts. Differential efferu of mlltation~ on ca.<;JlOfUrWn.. micafungin, and anidulat ltlgln susceptibility will be used to deduce the EC binding site. These data will be correlated with Fht topology, ftM PSM used to refine these models. (2) Examine the bflsis for intrinsic RES of selected fungal pathogens (ScedQsrorium , Cryp(QCQccus. and Aspergillus species) by hetaologous FKS expression in S. cerevisiae. Sequence aligrunents coupled with Aim 1 data will identity candidate RES-mediating regions. These will be used to generate fu l hybrids in S. ceevi.siae using the PSM method, followed. by tests ofEC susceptibility. Finally, PSM will be used to test the predicted roles of specific Fks residue;in intrinsic RES. These studies will accelerate the development of second generation ECs that provide a broader spectrum of protection from fungal pathogens.

Public Health Relevance

The echinocandins are an important new antifungal group which inhibit fungal ?-1,3-glucan synthase and hence cell wall synthesis. Reduced echinocandin susceptibility (RES) can develop in normally susceptible fungi such as Candida albicans by rare mutations of FKS genes encoding glucan synthase, but a greater clinical concern is the intrinsic RES exhibited by fungal pathogens including Fusarium, Scedosporium, Cryptococcus, Trichosporon, and zygomycete species. Our central hypothesis, that intrinsic RES is mediated by FKS sequence, will be tested by (1) mutationally defining the echinocandin target in Saccharomyces cerevisiae FKS1 and (2) heterologously expressing the FKS of fungal pathogens in S. cerevisiae;these studies should accelerate the development of second generation echinocandins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI075272-02
Application #
7849971
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Duncan, Rory A
Project Start
2009-06-01
Project End
2011-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
2
Fiscal Year
2010
Total Cost
$269,354
Indirect Cost
Name
Drexel University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
002604817
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Healey, Kelley R; Challa, Krishna K; Edlind, Thomas D et al. (2015) Sphingolipids mediate differential echinocandin susceptibility in Candida albicans and Aspergillus nidulans. Antimicrob Agents Chemother 59:3377-84
Katiyar, Santosh K; Alastruey-Izquierdo, Ana; Healey, Kelley R et al. (2012) Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance. Antimicrob Agents Chemother 56:6304-9
Johnson, Michael E; Edlind, Thomas D (2012) Topological and mutational analysis of Saccharomyces cerevisiae Fks1. Eukaryot Cell 11:952-60
Healey, Kelley R; Katiyar, Santosh K; Raj, Shriya et al. (2012) CRS-MIS in Candida glabrata: sphingolipids modulate echinocandin-Fks interaction. Mol Microbiol 86:303-13
Johnson, Michael E; Katiyar, Santosh K; Edlind, Thomas D (2011) New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species. Antimicrob Agents Chemother 55:3774-81
Healey, Kelley R; Katiyar, Santosh K; Castanheira, Mariana et al. (2011) Candida glabrata mutants demonstrating paradoxical reduced caspofungin susceptibility but increased micafungin susceptibility. Antimicrob Agents Chemother 55:3947-9