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Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

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Abstract:

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 °C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid – histidine – serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.Key words: extracellular serine protease, Monacrosporium microscaphoides, nematode-trapping fungus, nematocidal activity.

Afin de bien exploiter le potentiel de la lutte biologique contre les champignons nématophages, il est important de bien comprendre la base moléculaire du processus infectieux. Dans cet article, l'activité nématocide de plusieurs champignons nématophages a été examinée. Une sérine protéase neutre (identifiée Mlx) a été purifiée par chromatographie à partir d'un filtrat de culture de Monacrosporium microscaphoides. Cette protéase a pu immobiliser le nématode Penagrellus redivivus in vitro et dégrader sa cuticule purifiée, suggérant que Mlx serve de facteur de virulence durant l'infection. La caractérisation de la protéase purifiée a révélé une masse moléculaire d'approximativement 39 kDa, un point isoélectrique de 6,8 et une activité maximale à pH 9, 65 °C. Mlx a une large spécificité de substrat et hydrolyse des substrats protéiques parmi lesquels on retrouve la caséine, le lait écrémé, le collagène et l'albumine de sérum bovin. Le gène codant Mlx a aussi été cloné et la séquence de nucléotides, déterminée. La séquence en acides aminés déduite contient la triade catalytique conservée constituée d'un acide aspartique, d'une histidine et d'une sérine et démontre une forte similarité avec deux protéases pouvant dégrader la cuticule (PII et Aoz1) qui ont été purifiées du champignon nématophage Arthrobotrys oligospora. Jusqu'à présent, la recherche sur les mécanismes infectieux de champignons nématophages s'est concentrée sur A. oligospora. Cependant, on connaît peu de choses sur les autres champignons nématophages. Notre rapport est parmi les premiers à décrire la purification et le clonage d'une protéase infectieuse d'un champignon nématophage différent.Mots clés : sérine protéase extracellulaire, Monacrosporium microscaphoides, champignon nématophage, activité nématocide.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: February 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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