In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum.
A serine protease from the nematophagous fungus Monacrosporium thaumasium (NF34a) was purified, partially characterized and tested in vitro in control of the first larval stage of Angiostrongylus vasorum. NF34a grew in liquid culture medium, producing its crude extract that was purified by ion exchange chromatography. The fractions with high protease activity were collected in a pool, and elution of proteases was monitored by enzymatic assay and protein content. Purification steps were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protease activity was determined under different pH and temperature conditions, and the inhibitor effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) were assessed. In an experimental test, the infection process of NF34a on first-stage larvae of A. vasorum was investigated. A purified serine protease (Mt1) was identified, with an approximate molecular mass of 40 kDa and apparent homogeneity in SDS-PAGE, having optimal activity at pH 7.0 to 8.0 and temperature of 60°C. Mg(2+) and Zn(2+) partially inhibited the activity of Mt1 while PMSF inhibited it completely. Mt1 production was observed when NF34a was grown using first-stage larvae of A. vasorum as the only source of carbon and nitrogen. These results show that the enzyme may have a possible role in the infection process of the larvae. In the in vitro test of applicability against A. vasorum L(1), we observed a reduction in the number of larvae of 23.9% (p < 0.05) in the group treated with Mt1 compared with the control group. However, even this low reduction demonstrates that the Mt1 is important in the infection process.
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Cep: 3657000, Brazil.
Filippe E F
FE
Soares
Fabio R
FR
Braga
Jackson V
JV
Araújo
Walter
W
dos Santos Lima
Lanuze R
LR
Mozer
José H
JH
Queiróz
D000801:Angiostrongylus / Q000187:drug effects*; D000818:Animals; D000871:Anthelmintics / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D001681:Biological Assay; D002852:Chromatography, Ion Exchange / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D007814:Larva / Q000187:drug effects; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D016019:Survival Analysis; D013696:Temperature
D000871:Anthelmintics; D003470:Culture Media; D007477:Ions; D008670:Metals; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D057057:Serine Proteases