Extracellular serine proteases as virulence factors in nematophagous fungi: Molecular characterization and functional analysis of the PII protease in Arthrobotrys oligospora

Nematophagous fungi are parasites of nematodes, thus they have been considered and tested for biological control of parasitic nematodes, but so far with limited success. Due to the fact that the nematode cuticle is composed of proteins it is likely that fungal extracellular proteases are involved in the infection of nematodes. I have isolated a gene (designated PII) encoding an extracellular cuticle-degrading serine protease from the nematophagous fungi Arthrobotrys oligospora. The sequence of PII showed high homology to the subtilisin subclass including several functionally related fungal serine proteases. Experiments showed that the expression of PII was regulated by multiple regulatory circuits, including carbon and nitrogen repression. To further examine the function of PII as well as other putative virulence factors in A. oligospora, a transformation system was developed using hygromycin as selective marker. Using this system, PII deletion mutants, as well as PII over-expression mutants were constructed. One of the latter mutants infected significantly higher levels of nematodes compared to the wild-type, demonstrating that PII is a virulence factor in A. oligospora. The PII disrupted mutants retained virulence suggesting that there are other proteases involved in the infection. A major role of PII during infection is probably to degrade the cuticle and host tissues, providing nutrients for development of infections structures or, alternatively, vegetative mycelium. To be able to investigate the biochemical properties of PII in more detail without interference from other functionally related enzymes, PII was over-expressed in a heterologous host (Aspergillus niger), at a level of almost 100-fold higher than in A. oligospora. All measured enzymatic properties in the recombinant PII were similar to those of native PII. The thesis demonstrates that genetical engineering of fungal proteases has a potential to improve the virulence of nematophagous fungi, information that in the future can be used for developing more efficient strains of fungi for biological control of parasitic nematodes.