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.
de Gouvêa
Paula Fagundes
PF
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Cafe S/N, CEP 14040-903 Ribeirão Preto, São Paulo, Brazil.
Soriani
Frederico Marianetti
FM
Malavazi
Iran
I
Savoldi
Marcela
M
Goldman
Maria Helena de Souza
MH
Loss
Omar
O
Bignell
Elaine
E
da Silva Ferreira
Márcia Eliana
ME
Goldman
Gustavo Henrique
GH
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