Abstract
ABSTRACT A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.
Highlights
More than 100 families of metalloproteases (MPs) have been identified from different organisms and curated at the MEROPS database [1]
For seven M35 MPs encoded in M. robertsii, MrM35-2 and MrM35-4 are closely related to each other and clustered together (SF8) with the Magnaporthe Avr-Pita effector [8]
Apoptotic cells were counted after trypan blue staining. **, t-test, P = 0.0011. (d) Survival of the wild-type, PPO1Δ, PPO2Δ, and PPO1ΔPPO2Δ mutants of D. melanogaster after topical infection with the WT and different mutants of M. robertsii
Summary
More than 100 families of metalloproteases (MPs) have been identified from different organisms and curated at the MEROPS database [1]. A M10 family AprA is a virulence factor of Pseudomonas entomophila during bacterial infection of Drosophila melanogaster by cleavage and maturation of a bacterial pore-forming toxin [4,5]. AprA is required for bacterial infection of the bean bugs (Riptortus pedestris) by suppressing host cellular and humoral immunities [6]. Different MPs have been characterized in pathogenic fungi with virulence contributions against different hosts. In fish pathogenic bacterium Aeromonas salmonicida, a M35 MP AsaP1 is found indispensable for bacterial virulence against fishes [9].
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