Cloning and sequencing of partial plb2 gene of Aspergillus fumigatus

ABSTRACT

Aspergillus species are primarily opportunistic pathogens and with a few exceptions cause invasive disease only in an immunocompromised hosts during last two decades, the importance of fungal disease has been increased dramatically due to an increased occurrence of tuberculosis, chemotherapy-induced neutropenia, AIDS, open chest surgery and use of antibiotics and immunosuppressive therapy. The production of extracellular phospholipases by pathogenic fungi such as A. fumigatus are involved in the degradation of target cell membrane phospholipids in invaded tissues. Using degenerate primers based on homologous amino acid sequences of fungal phospholipase B [PLB] gene, PCR products of 542 was generated which was the predicted size. Sequence analysis revealed sequence had a high homology to published PLB sequences sharing highest homology to Aspergillus oryzae PLB [62% identity]. Inverse PCR was used to attempt to clone the entire plb2 genes. Initially, genomic DNA was digested with selected restriction enzymes and self ligated with T4 DNA ligase to give circular DNA for inverse PCR [IPCR] for A. fumigatus plb2. IPCR with Xho I self-ligated digest gave product of 1.2 Kb for plb2 and was found to encode the 3' ends of gene and using CAP assembly software this sequence was assembled with 542 bp sequence. BLST X and Gene Finder analysis revealed that the first 542 bp of assembled total 1529 bp [after deletion of similar bp from both sequences] encoded the 3' end of plb2 gene. The sequence availability of partial afplb2 gene and after that full sequence of this gene represents a major breakthrough in the study of this opportunistic pathogen and function of plb gene in pathogenesis of A. fumigatus. Also phylogenic tree analysis of afplb2 gene shows that PLBs and potential PLB analogues are contained in a large cluster of the PLB family. A. fumigatus are more closely relaed to A. oryzae, A. niger, P. notatum and N. crassa PLB than other fungi