Functional characterization of the Aspergillus fumigatusPHO80 homologue.
Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P(i)) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P(i) at normal or high external P(i) concentrations) and a high-affinity (activated in response to P(i) starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB(PHO80). We show that the DeltaphoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and DeltaphoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the DeltaphoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1mM P(i). Epifluorescence microscopy revealed accumulation of poly-phosphate in DeltaphoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) 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, DeltaphoB(PHO80) and DeltaphoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis.
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Cafe S/N, CEP 14040-903 Ribeirão Preto, São Paulo, Brazil.
Paula Fagundes
PF
de Gouvêa
Frederico Marianetti
FM
Soriani
Iran
I
Malavazi
Marcela
M
Savoldi
Maria Helena de Souza
MH
Goldman
Omar
O
Loss
Elaine
E
Bignell
Márcia Eliana
ME
da Silva Ferreira
Gustavo Henrique
GH
Goldman
D000818:Animals; D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008297:Male; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010710:Phosphates / Q000378:metabolism*; D014774:Virulence
D005656:Fungal Proteins; D010710:Phosphates; D016572:Cyclosporine; D017868:Cyclic AMP-Dependent Protein Kinases; D018844:Cyclin-Dependent Kinases; D002118:Calcium