Understanding the pathogenic mechanisms of Aspergillus flavus in human mycotic keratitis using high efficiency gene targeting system

Mohammed Razeeth. S*. Jeyalakshmi K, Lalitha P, Venkatesh Prajna N, and Dharmalingam K

Author address: 

Department of proteomics, Aravind Medical Research foundation, Aravind Eye Hospital, Anna Nagar, Madurai-625 020

Abstract: 

Fungal keratitis is an inflammatory disease of cornea resulting from infection by a fungus. The fungus has the ability to break through the multiple layers of cornea and enter into the anterior chamber of cornea. Fungal keratitis can lead to loss in visual acuity and in extreme cases even to blindness. In order to understand the pathogenic mechanisms employed by the invading fungus we examined the exoproteome of the causative fungus which would give an insight of the proteins involved in the early events in infection process. Exoproteins being the early proteins elaborated by the fungus study of these will allow us to understand the pathogenic mechanisms. In the present study, compared the exoproteome of ATCC and clinical strains isolated from keratitis patients were compared. The cultures were grown on solid-state fermentation and the secreted proteins were extracted. SDS-PAGE analysis showed up regulation of a 37KDaproteinin clinical isolates. Mass spectrometric analysis ofthe extracted proteins identified the protein as alkaline protease. Subsequently, two-dimensional and western blot analysis confirmed the identity of the protein. In order to further explore the role of this protein in pathogenesis we used gene-targeting approach to knock out the gene encoding the protease. We have constructed a replacement vector for alkaline protease gene and used for transforming the conidia of Aspergillus flavus A1421 ApyrGAKu7 0. The alk disruption vector was contracted in two steps. 0.6 kb alk coding and non-coding region at 5' end was amplified by PCR using primers alk 5Sp and alk 5Sa. All PCR fragments were digested with Sph I and Sa II was cloned in to respective site of pPG535. Then 0.7 Kb alk coding and noncoding region at 3' end was amplified by PCR using primers alk 3Ba and alk 3Sc. PCR fragments were digested with BamHl and Sacl the fragments was cloned in to vector obtained from previous step. Disruption vector was linearized by digest with Hindlll prior to fugal transformation. 1 p.g of DNAused for transformation. Transformation was through electroporation. Transformed colonies were selected based on uracil auxotrophy. Exoproteome of this isolate was examined. Interestingly, the knockout strain showed completely altered protein profile. In western blot alkaline protease was not identified from the knockout strain. Protease activity of knockout strain and parental strain was examined by Azo-Casein assay. The knockout strain showed high protease activity when compared to parental strain. ’Corresponding author E-mail:[email protected]
2014

abstract No: 

OP-018

Full conference title: 

Society for Indian Human and Animal Mycologists 2014
    • SIHAM