Aspergillus fumigatus is an opportunistic pathogen predominantly affecting immunocompromised individuals, resulting in pulmonary illnesses such as Invasive Aspergillosis. Sequencing of the genome has led to an increased understanding of the organism; however the functions of many genes remain unknown. A putative translation elongation factor 1Bγ (EF1Bγ , termed elfA; 750 bp) is expressed, and exhibits glutathione s-transferase activity, in A. fumigatus . Normally, EF1Bγ plays a key role in the elongation step of protein synthesis. Our hypothesis is that elfA may also play a role in regulating the cellular redox state adjacent to the ribosome during protein synthesis. Consequently, elfA was disrupted in A. fumigatus ATCC46645 (wild-type) using a bipartite construct containing overlapping fragments of a pyrithiamine resistance gene (ptrA). The elfA mutant (916;elfA) was complemented using a hygromycin resistance marker (hph). Southern Blot analysis was used to confirm the generation of 916;elfA and the complemented strain. RT-PCR confirmed the expression of elfA in wild-type and complemented strains, and absence of expression in 916;elfA. The availability of the mutant has facilitated phenotypic analysis of elfA functionality. A. fumigatus wild-type and 916;elfA were grown on AMM plates with the oxidant H2O2 (1 - 5 mM), voriconazole (0.25 - 1 Âµg/ml), and the thiol-reactive reagent, 4,4’-dithiodipyridine (3 - 7.5 ÂµM). At 37Â°C, the elfA mutant was significantly more sensitive (p=0.0003) to H2O2 than wild-type. However, 916;elfA was significantly less sensitive (p=0.0251) to voriconazole than wild-type. At 37Â°C, the 916;elfA was significantly more sensitive (p=0.0056) to 4,4’-dithiodipyridine than wild-type. These results implicate elfA in the oxidative stress response in A. fumigatus and also strongly indicate that elfA may play a role in the sensitisation of A. fumigatus to voriconazole. Global proteomic studies are currently underway using 2D-PAGE and MALDI-MS to explore alterations in the proteome consequential to elfA disruption with a view to gaining further insight into the function of elfA in A. fumigatus.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)