Functional Analysis of Penicillium marneffei sakA and atfA Genes Involved in Oxidative Stress Response

Abstract

Penicillium marneffei, the pathogenic dimorphic fungus is a causative agent of a fatal systemic disease, penicilliosis marneffei, in immunocompromised hosts especially in HIV patients. For growth and survival outside and inside host cells, this fungus has to adapt to environmental stresses including heat, osmotic, oxidative or nutrient stress and this adaptation requires stress signaling pathways and regulation of gene expression under various kinds of stresses. However, the strategies used by this fungus in response to environmental stresses are still unclear. In the present study, the stress activated kinase (sakA) gene encoding the mitogen activated protein (MAP) kinase and activating transcription factor (atfA) gene encoding bZip-type transcription factor of P. marneffei were characterized. Results demonstrated that the P. marneffei sakA gene encoded a putative protein containing TXY phosphorylation lip found in the stress high osmolarity glycerol 1 (Hog1)/Spc1/p38 MAPK family and P. marneffei atfA gene encoded a putative protein including conserved basic-leucine zipper (bZip) domain found in the bZip transcription factor family. RT-PCR revealed the increase of sakA gene expression in conidia under treatment with 1 mM hydrogen peroxide and heat at 39ºC for 1 h, whereas the expression of atfA gene did not increase in any conditions. To determine functions of these genes, sakA and atfA mutant strains were constructed using the modified split marker recombination method. The phenotypes and susceptibility to varieties of stresses including osmotic, oxidative, heat, UV and cell wall stresses of the mutant strains were compared with the wild type and the complemented strains. The results demonstrated that sakA gene but not atfA gene played a role in conidiation (asexual development) and yeast cell transition of P. marneffei in vitro and inside mouse and human macrophages. In addition, neither sakA nor atfA was involved in osmotic and UV stress responses. For oxidative stress response, sakA gene participated in response of P. marneffei conidia against hydrogen peroxide and tert-butyl hydroperoxide but not menadione, whereas atfA gene was only associated with response to tert-butyl hydroperoxide. For cell wall integrity evaluation, the results showed that sakA gene was involved in chitin deposition along the hyphae of P. marneffei but was not required for response against cell wall stress agent calcofluor white at 25ºC. However, both sakA and atfA genes played a role in response to stress from sodium dodecyl sulphate which is a membrane destabilized agent. For heat stress, sakA gene was necessary for survival of P. marneffei conidia at 39ºC, whereas atfA gene was not involved in growth of the conidia at both 39ºC and 42ºC. Nevertheless, quantitative real-time PCR revealed that heat stress condition at 42ºC for 20 min was able to activate the mRNA expression levels of both sakA and atfA genes. In macrophage infection experiment, recovery of sakA and atfA mutant conidia from the infected human or mouse macrophages was significantly decreased compared to those of wild type and complemented strains. The results from this study indicated that the sakA and atfA genes were required by P. marneffei under specific stress conditions and might be necessary for fighting against host immune cells during the initiation of infection.