System of inorganic phosphate acquisition in the human pathogen fungi Aspergillus fumigatus

Author(s)

Paula Fagundes de Gouvêa

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2009-03-27

Examining board members

Gustavo Henrique Goldman; Paulo Sergio Rodrigues Coelho; Marcia Eliana da Silva Ferreira

• Advisor: Gustavo Henrique Goldman

Abstract

Environmental sensing and retrieval of nutrients from the environment are associated with growth of A. fumigatus in inhospitable environments. Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80, PHO84 and PHO85 homologues, here denominaded phoBPHO80, phoDPHO84 and phoAPHO85, respectively. We show that the phoBPHO80 mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoBPHO80, calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and phoBPHO80 mutant cells identify Afu4g03610 (phoDPHO84), Afu7g06350 (phoEPHO89), Afu4g06020 (phoCPHO81), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the phoBPHO80 mutant background and under phosphate-limiting conditions of 0.1 mM Pi. Epi-fluorescence microscopy revealed accumulation of poly-phosphate in phoBPHO80 vacuoles, which was independent of extracellular phosphate concentration. We also tried to isolate the phoAPHO85 deletion strain without succes after several times what raises the interesting possibility that the phoAPHO85 null mutant might be essential in Aspergillus fumigatus. Surprisingly, phoDPHO84 deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, phoBPHO80 and phoDPHO84 mutant are fully virulent in a murine low dose model for invasive aspergillosis. (AU)