Cas5, Upc2, and Rpn4 Influence the Fungistatic Activity of Fluconazole Against Candida albicans

Ref ID: 18766

Author:

E. M. Vasicek, BS – Graduate Student, E. L. Berkow, MS – Graduate Student, K. S. Barker, PhD – Assistant Professor, P. D. Rogers, PharmD, PhD – Professor;

Author address:

Univ. of Tennessee Hlth. Sci. Ctr., Memphis, TN.

Full conference title:

52nd Annual ICAAC

Date: 9 September 2014

Abstract:

Background: The azole antifungal agents such as fluconazole (FCZ) exhibit fungistatic activity against Candida albicans. Strategies to render the azoles fungicidal are therapeutically appealing for the management of Candida infections. In an effort to identify transcriptional pathways involved in this fungistatic activity, we sought to identify transcription factors (TFs) essential for this process. Methods: FCZ MICs and MFCs were determined against a collection of 165 mutant strains of C. albicans disrupted for genes encoding TFs (Homann et al., PLoS Genet. 2009;5:e1000783). TF genes of interest (GOI) exhibited marked reduction in MFC in both RPMI and YPD media independent of any noted growth defect in medium alone as compared to wild-type (wt). GOIs were disrupted independently in SC5314 using the SAT1 flipper method and were assessed by broth microdilution MIC/MFC testing, E-test, antifungal spot assays, growth curves, and time-kill analysis. Results: We identified 3 TF mutants that met criteria for a GOI: CAS5, RPN4, and UPC2. CAS5 and RPN4 disruption had minimal impact on FCZ MIC by broth microdilution, E-test, and spot assay at 24 h. Disruption of UPC2 resulted in a marked reduction in MIC by all methods. The MFC at 24 h for wt was >64 μg/mL whereas cas5916;/916;, rpn4916;/916; and upc2916;/916; were 1, 16, and 0.25 μg/mL respectively. At 48 h, confluent growth was observed by E-test for wt and rpn4916;/916;, whereas a clear zone of inhibition was observed for cas5916;/916; and upc2916;/916;. Likewise, in MIC plates at 72 hr wt and rpn4916;/916; were able to grow in all concentrations of FCZ tested whereas cas5916;/916; and upc2916;/916; were not. Growth was reduced in the presence of FCZ for cas5916;/916; and upc2916;/916; as compared to wt, and time-kill analysis revealed a fungicidal effect of FCZ against both at 10 μg/mL. All phenotypes were reverted by reintegration of one allele of the disrupted gene. Conclusions: These data suggest that Cas5, Upc2, and to a lesser extent Rpn4 all regulate transcriptional networks that influence fungistatic activity of FCZ against C. albicans. Further delineation of these transcriptional networks may identify potential targets for co-therapeutic strategies to impart fungicidal activity to the azole class of antifungals.

Abstract Number: M-971

Conference Year: 2012

Link to conference website: NULL

New link: NULL

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