Control  of azole resistance Aspergillus fumigatus by using of RNAi based on cyp 51A gene

Ref ID: 19552

Author:

B Mousavi1*, MT Hedayati1, H Badali1, L Teimoori2, SM Seyedmousavi3, A Alizadeh4

Author address:

1Medical Mycology and Parasitology, Invasive Fungi Research Center (IFRC), Mazandaran University of
Medical Sciences, Sari, Iran
2Molecular Medicine, Biotechnology Research Center , Pasteur Institute, Tehran, Iran
3Department of Medical Microbiolo

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Drug resistant in environmental and clinical isolates of Aspergillus has revealed in recent decades has
been detected especially in A. fumigatus. Two important factors which leads to route of action and
azole resistance mechanism is either long term exposure to azoles in patients with aspergillosis
or confront to azole fungicides in agriculture fields. Anyhow the evolution of drug resistance is more
likely to proceed by the mutations in amino acids or less by MDR genes overexpressed. Majority
cases of azole resistance A. fumigatus are related with mutations in the azole drug target enzyme
encoded by the cyp51A gene. Therefore, here we evaluated RNA silencing technology for decreasing
of resitancy to azole treatment in patients with aspergillosis.
Methods:
siRNA sequences was designed to target the mRNA sequence of the cyp51A . The 21-nucleotide
siRNA was designed on the basis of the cDNA sequence of the Cyp51A gene of A. fumigatus.
The sense and antisense sequences were 5’-UGGCAAGCACAAGGACGUUAA-3’ and
5’FAM8209;UUAACGUCCUUGUGCUUGCCA-3’ respectively. Also a scramble sequence which is
unrelated and random siRNA of the mentioned target with the sense and antisense oligonocleotides
5’-GAUGGCAUACCUAGGAGAACA-3’ and 5’-CUACCGUAUGGAUCCUCUUGU-3’ was
obtained from a distinct site and synthesized as a negative control. The antisense strand of siRNA
was labled with the FAM fluorescent dye to monitor the entrance of siRNA in mycelia. To investigate
cyp51A gene silencing in siRNA treated germinated conidia (15, 20, 25 and 50nM), azole resistant
Aspergillus was cultured on Czapek-dox broth, then quantitative changes in expression of the
cyp51A gene were analyzed by measuring the cognate cyp51A mRNA level by use of a RT-PCR
assay. Statistical analysis was performed by using of R software version 3.0.1.
Results:
With fluorescence microscopy, it was approximately appraised which all mycelia with used
concentrations had been transfected by siRNA successfully. Compared with the positive control,
expression of the cyp51A gene was significantly reduced by siRNA at concentrations of 50 nM
(P=0.05). In addition, siRNA at mentioned concentration, revealed to decrease MIC effectively.
Conclusion:
The correlations between changes in cyp51A gene expression and MIC suggests RNA silencing
technology could be used as a efficient means for control of azole resistancy in patients with
aspergillosis.
Keywords:
Azole resistance, Aspergillus fumigatus, RNAi, cyp51A gene

Abstract Number: 79

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

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