Background: Mechanisms unrelated to cyp51 mutations have been demonstrated to be responsible for triazole resistance in clinical isolates of A. fumigatus. Studies on signaling pathways and their role if any in triazole reisstance in Aspergillus species is limited. Protein kinase C is a central component of the cyclic AMP signaling cascade in the human opportunistic mold A. flavus that regulates cellular processes by phosphorylation of specific proteins. It has been shown to be involved in both conidiation/pigmentation pathways (via alb1 gene) in fungi. Recent studies have also evaluated the role of these pathways in virulence in A. fumigatus. However the role of pkaC and alb1 gene mutations in triazole resistance in A. flavus is unknown. Aim: To characterize and study the expression of pkaC and alb1 genes in WT and pigmentless, non-conidiating VCZ-R (MIC 4 mcg/ml) clinical isolate of A. flavus isolate that lacks cyp51A mutation. Methods: Fresh cultures of WT and VCZ-R A. flavus were used for DNA and RNA isolation (Qiagen kit). Gene specific primers were designed and used to amplify the complete transcriptional unit of pkaC and alb1 genes using standard PCR and qRT-PCR (Taqman probes) conditions as appropriate. The amino acid sequences of pkaC and alb1 genes in WT and VCZ-R A. flavus were deduced from the nucleotide sequence and compared. Any change in expression of pkaC was tested using qRT-PCR using 18sRNA as the housekeeping gene. Results: We have characterized the pkaC gene of A. flavus for the first time in an albino clinical isolate of A. flavus that is VCZ-R (MIC 4 mcg/ml). The pkaC gene in WT A. flavus demonstrated 99% homology (3 exons, 3 introns, 1158bp, 385aa) with the published NRRL3357 pkaC gene sequence. No mutations in pkaC were demonstrated in VCZ-R albino A. flavus although there were amino acid mutations involving the alb1 or the pigmentation gene. Differential expression of pkaC was observed in various VCZ-R isolates of A. flavus. Discussion: Lack of conidiation and pigmentation were associated with alb1 but not pkaC mutations in VCZ-R albino A. flavus. However differential expression of pkaC was demonstrated in pigmentless, non-conidiating VCZ-R A. flavus. Both pkaC and alb1 genes are involved in the conidiation and pigmentation pathways respectively and may contribute to triazole resistance in VCZ-R A. flavus. The contribution of the protein kinase pathway to conidiation/pigmentation and triazole resistance in A. flavus needs to be further explored. The virulence potential of these albino mutants of A. flavus needs to be investigated using an in vivo model.
Full conference title:
- AAA 5th (2012)