Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence.
OBJECTIVE
To determine the mechanisms responsible for fluconazole resistance in two Candida albicans isolates (CAAL2 and CAAL76) recovered from two hospitalized patients after fluconazole prophylaxis.


METHODS
MICs of fluconazole and voriconazole were determined by the broth microdilution method (CLSI M27-A3), and by Etest(®) for amphotericin B. RNA expression levels of CDR1, MDR1 and ERG11 were determined by RT-PCR. Mutations in ERG11 and ERG3 were investigated by amplification and sequencing. Sterol membrane profiles were determined by gas chromatography-mass spectrometry (GC-MS). In vivo virulence was determined in a murine model of invasive candidiasis.


RESULTS
Both isolates displayed azole cross-resistance and reduced susceptibility to amphotericin B, and are novel Δ(5,6)-desaturase (Erg3p) mutants. CAAL2 harbours a new amino acid substitution (L193R), whereas a 13 bp deletion leading to a truncated Erg3p (Δ366-378) was found in CAAL76. Both genetic alterations impaired Erg3p function as shown by GC-MS in these isolates (ergosterol content below 10%, and accumulation of ergosta-7,22-dienol above 40%). In vivo, in a murine model of invasive candidiasis, both CAAL2 and CAAL76 exhibited a significant trend toward reduced virulence, which seems to be linked to a reduced capacity for hyphal growth.


CONCLUSIONS
These findings demonstrate the critical role of residue 193 in Erg3p function and azole resistance. We suggest that this attenuated in vivo virulence phenotype could be linked to lower potential for hyphal growth. Taken together, our findings highlight the fact that erg3 mutants must be considered in future studies aiming at investigating azole antifungal drug resistance.
Département de Parasitologie et Mycologie Médicale,  Université de Nantes, Nantes Atlantique Universités, EA1155-IICiMed, Faculté de Pharmacie, Nantes,  France.
Florent
F
Morio
Fabrice
F
Pagniez
Claire
C
Lacroix
Michel
M
Miegeville
Patrice
P
Le Pape
D000328:Adult; D019943:Amino Acid Substitution; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity; D002177:Candidiasis / Q000382:microbiology; D002462:Cell Membrane / Q000737:chemistry; D018890:Chemoprevention / Q000379:methods; D025141:Drug Resistance, Fungal; D005260:Female; D015725:Fluconazole / Q000494:pharmacology* / Q000627:therapeutic use; D008401:Gas Chromatography-Mass Spectrometry; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D013261:Sterols / Q000032:analysis; D014230:Triazoles / Q000494:pharmacology; D014774:Virulence; D065819:Voriconazole
D000935:Antifungal Agents; D050505:Mutant Proteins; D011743:Pyrimidines; D013261:Sterols; D014230:Triazoles; D015725:Fluconazole; D010088:Oxidoreductases; C039306:sterol delta-5 desaturase; D065819:Voriconazole