Characterization and functional analysis of the beta-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis.
The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, beta-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1Delta mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a beta-1,3-glucan elongase capable of mediating fungal cell wall integrity.
Castro
Nadya da Silva
Nda S
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
de Castro
Kelly Pacheco
KP
Orlandi
Ivan
I
Feitosa
Luciano dos Santos
Ldos S
Rosa e Silva
Lívia Kmetzsch
LK
Vainstein
Marilene Henning
MH
Báo
Sônia Nair
SN
Vai
Marina
M
Soares
Célia Maria de Almeida
CM
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D018628:Gene Dosage; D015870:Gene Expression; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D025282:Mycelium / Q000737:chemistry; D010228:Paracoccidioides / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015003:Yeasts / Q000737:chemistry
D018076:DNA, Complementary; C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase