646. Mechanisms of Azole Resistance in Cryptococcus gattii
Session: Abstracts: Mycology
Friday, October 22, 2010
Background:

Patients with serious Cryptococcus gattii (Cg) infections in the Pacific Northwest (NW) often respond poorly to azoles.  The frequencies and mechanisms of azole resistance in Cg have not been studied.

Methods: and Results:

The MICs for several azoles were approx 2-fold higher in NW Cg (n=25) than in Cg from other areas (non-NW, n=34) or in C. neoformans (Cn, n=20), but there were no differences in AmB MICs.  In azole-unexposed cells, ERG11 mRNA levels did not differ between the 3 groups and did not correlate with azole MICs within any group.  ERG11 cDNAs from 2 NW Cg and 2 Cn (serotypes A and D) were cloned by RT-PCR and complemented a Saccharomyces cerevisiae (Sc) ERG11(tet off) strain, but these transformants’ fluconazole (flu) MICs did not differ.  Intracellular [3H]-flu did not differ between the 3 groups, but intracellular [3H]-flu inversely correlated with flu MICs in the NW Cg, but not the other groups.  Since this suggested that plasma membrane (PM) efflux pumps may contribute to azole resistance in Cg, we searched the Cg genome database and found 7 homologs of PM azole efflux pumps from other fungi.  Four of these were expressed in Cg, and Sc AD1-8u (which lacks 7 PM ABC transporters) expressing 3 corresponding cDNAs (Cg homologs of Cn MDR1, Cn AFR1 and Candida albicans CDR1/CDR2 [now named Cg AFR2]) had higher flu MICs and lower intracellular [3H]-flu than did empty-vector controls.  Lastly, post-Golgi secretory vesicles (PGVs) isolated from lysed spheroplasts of Sc AD1-8u sec6-4(ts) mutants expressing Cg MDR1, AFR1 and AFR2 cDNAs accumulated more [3H]-flu than did PGVs from empty-vector controls.

Conclusion:

We conclude that (i) neither ERG11 overexpression nor inherent resistance of Erg11p to azoles can explain the high azole MICs observed in NW Cg and (ii) Cg MDR1, AFR1 and/or AFR2 encode ABC transporters that can pump [3H]-flu out of whole Sc cells and into the lumens of Sc PGVs.  Future studies will examine (i) the catalytic properties of Cg Mdr1p, Afr1p and Afr2p (ie, Km’s, Vmax’s, energy requirements, substrate specificities, and inhibitor profiles), (ii) MDR1, AFR1 and AFR2 expression in azole-susceptible and resistant Cg, and (iii) the phenotypic consequences of the mdr1, afr1 and afr2 null mutations in Cg.


Subject Category: M. Mycology including clinical and basic studies of fungal infections

Speakers:
Luiz R. Basso Jr., PhD , Medicine/Infectious Diseases, Oregon Health & Science University, Portland, OR
Charles Gast, BA , Medicine/Infectious Diseases, Oregon Health & Science University, Portland, OR
Brian Wong, MD , Medicine/Infectious Diseases, Oregon Health & Science University, Portland, OR

Disclosures:

L. R. Basso Jr., None

C. Gast, None

B. Wong, None

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