Overexpression of Four Novel Multi-Drug Resistance (MDR) Transporter Genes Encoding ABC or MFS Class Efflux Proteins Contribute to Voriconazole Resistance (VCZ-R) in Aspergillus flavus

Ref ID: 15149

Author:

S. KRISHNAN-NATESAN, K. JOST, P. CHANDRASEKAR

Author address:

1Wayne State Univ., Detroit, MI, 2John D. Dingell VA Med. Ctr., Detroit, MI.

Full conference title:

50th Annual ICAAC

Date: 12 September 2014

Abstract:

Background: Invasive aspergillosis remains a life threatening infection; clinical failures may be attributed to antifungal drug resistance. Research on Aspergillus efflux pumps (EPs) is scant. Aim: To develop a Rhodamine-6G based spectrophotometric assay for drug efflux and to evaluate overexpression of MDR-EPs in VCZ-R A. flavus that lack cyp51A mutation. Methods: VCZ-R A. flavus (n=10; MIC 2-16 µg/ml, laboratory-selected) were screened for ABC: ATP Binding Cassette [± CCCP (8 mcg/ml)] and MFS: Major Facilitator Superfamily MFS [± glucose (1M)] EP overexpression using a R6G-FA. Isolates that showed efflux (n=5) were selected, grown in the presence and absence of VCZ (n=10) and RNA isolated. Four novel EPs (ABC1, ATRF, MDR4 and MFS1) were identified in A flavus (NRRL 3357), amplified using Taqman-qRT-PCR; results for parent vs VCZ-R A. flavus (±VCZ) were compared. Isolates were also tested for cyp51A mutations. Results: None of VCZ-R A. flavus tested had cyp51A mutation. Inhibition (ABC-EP) or excitation (MFS-EP) of R6G efflux was demonstrated with CCCP and glucose respectively in 5/10 VCZ-R A. flavus. Constitutive overexpression (>7-fold) of ATRF (n=4), ABC1 (n=4) and MDR.4 (n=1) was demonstrated in VCZ-R A. flavus; inducible overexpression (>10-fold) of ATRF and ABC1 was seen in the VCZ-S parent; ABC1, MFS and MDR 4 were induced >100-fold in 1/5 VCZ-R A flavus; MFS was downregulated in 2/5 VCZ-R A flavus. Conclusion: Screening using R6G assay detects possible upregulation of ABC and MFS EPs; EP inhibition with CCCP suggests that A. flavus MDR4, ATRF and ABC1 belong to ABC-family of EP. VCZ is a substrate for both ABC and MFS class EPs. Multiple EPs are induced on exposure to sub-MIC of VCZ. Constitutive upregulation of several MDR-EPs in the absence cyp51A mutation may play a crucial role in phenotypic VCZ-R in A. flavus.

Abstract Number: M-626

Conference Year: 2010

Link to conference website: NULL

New link: NULL


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