Memoria CD.indd - ISHAM

4-11β1,3-glucan synthase: Recombinant protein, cytolocalization, activity and studies understress conditionP. K. Tomazett 1 , Félix, C. R 2 ., Barbosa, M. S 1 ., Santana, J. M 3 ., Faria, F. P 1 ., Báo, S. N 4 ., Soares, C. M. A 1 ., Pereira, M 1 .1 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia-GO-Brazil. 2 Laboratóriode Enzimologia, Instituto de Ciências Biológicas, Universidade de Brasília, Goiânia-GO-Brazil. 3 Faculdade de Medicina, Universidade deBrasília, Goiânia-GO-Brazil. 4 Laboratório de Microscopia Eletrônica, Instituto de Ciências Biológicas, Universidade de Brasília, Goiânia-GO-Brazil. e-mail: patriciakt@hotmail.comThe cell wall of fungi is an essential and antigenic structure. The β-1,3-glucan polymer possibly givesshape to the cell and its synthesis, which occurs at the plasma membrane, may be the result of the activityof β-1,3-glucan synthase. In P. brasiliensis just one gene homologue of β-1,3-glucan synthase (PbFKS1) hasbeen characterized. Here, the catalytic subunit of glucan synthase (PbFKS1c) was used to obtain a recombinantprotein in Escherichia coli. The pGEX-4T-3 vector was used to produce the recombinant protein in fusion withGlutathione S-transferase (GST) in its active form. The enzymatic assay of the recombinant and native glucansynthase was performed using a radioisotopic method where the substrate UDP-glucose is radioactively labeled.The purified recombinant PbFKS1c was used to generate specific rabbit polyclonal serum. This antibody was usedto perform an immunocitolocalization of glucan synthase in P. brasiliensis yeast cells. Glucan synthase activity iscurrently assayed by the radioisotopic method. However, this method is expensive and requires special handlingand disposal methods for radioactive wastes. Based in a method described to soybean seeds galactinol synthasewe have adapted a new colorimetric method to determine the glucan synthase activity. The colorimetric methodhas been standardized with the native glucan synthase. The reaction time, protein concentration and substrateconcentration have already been determined. The colorimetric assay is still being tested to the recombinant proteinPbFKS1c. The growth of P. brasiliensis was evaluated under stress conditions to determine the concentration inwhich the fungus is sensible to the stressors agents calcofluor white, congo red, SDS, NaCl, KCl and sorbitol,including cell wall damage and osmotic stress. By using the concentration in which the fungus is visually sensibleto those agents, the transcript level glucan synthase was evaluated by real time RT-PCR, and the enzymaticactivity was performed by using colorimetric method.4-12The α-amylase (amy1) gene in Paracoccidioides brasiliensis, identification andsemiquantitative expressionE. Camacho, Niño-Vega, G., and San-Blas, G.Instituto Venezolano de Investigaciones Científicas, Centro de Microbiología y Biología Celular, Apartado 20632, Caracas 1020A,Venezuela. e-mail: ecamacho@ivic.veIn Paracoccidioides brasiliensis, an α-1,3-glucan is present as the outer layer of the cell wall in the yeastlikephase, a polysacharide that is absent in the mycelial phase. α1,3-Glucan has been proposed as a virulence factornot only in P. brasiliensis but also in Blastomyces dermatitidis and Histoplasma capsulatum. An α1,4-amylase isessential for the synthesis of cell wall α1,3-glucan and expression of the virulence in H. capsulatum. With theaim of exploring the possibility of similar functions in P. brasiliensis, we identified, isolated and characterized itsα-amylase (amy) gene.P. brasiliensis IVIC Pb73 (ATCC 32071) was grown in PYG medium, at 23ºC (mycelial phase, M) or 37ºC(yeastlike phase, Y). Genomic DNA was extracted from freeze-dried M cultures. Total RNA was extracted withTRIzol from frozen Y cells. To amplify the gene, degenerate and gene-specific oligonucleotides were sequentiallyused, the latter by means of a RACE reaction. Semiquantitative RT-PCR was applied for gene expression, using 3µg total RNA as template, in 10-30 cycles, to determine the exponential phase in the expression of P. brasiliensisamy1 and 18S genes.A gene with high identity with H. capsulatum α-1,4-amylase, expressed preferentially in the pathogenic Yphase, was identified. In silico amino acid analysis of the deduced protein led to the identification of all fourconserved regions of the αamylase family, the critical moieties for biological activity and amino acids associatedwith the specificity to glucosidic α-1,4-linkages. Currently, the participation of such gene in the remodeling of thecell wall architecture in P. brasiliensis, pathogenic Y phase is under study.Acknowledgements: To FONACIT (Caracas, Venezuela) and ICGEB (Trieste, Italy) for partial financial support.170