Characterization of Batrachochytrium dendrobatidis motility and elastolytic enzyme activity

Abstract

Batrachochytrium dendrobatidis (Bd) is a chytrid fungus pathogenic to amphibians. Infections with this organism result in chytridiomycosis, a disease that is implicated in mass population declines and extinctions of amphibians around the globe. The infective stage of the fungus is a motile zoospore. The zoospore can infect the keratinized tissues of susceptible amphibians, such as the mouthparts of tadpoles and the epidermis of adults. This study investigated the ability of the fungus to exhibit positive movement towards a variety of attractants including nutrients and host-relevant components. At concentrations of 0.2%, growth media components such as glucose, lactose, gelatin hydrolysate, and casein, elicited a positive response in zoospores when compared to water. Zoospores were also attracted to the host-relevant protein, keratin, at a concentration of 2%. Additionally, 1 !M epinephrine also induced positive movement of zoospores. Of the two predominant amino acids found in keratin that were tested (cysteine and glutamic acid), zoospores showed a preference for the cysteine component. Zoospores also swam towards the amino acid glycine, a predominant component of gelatin hydrolysate. The fungus also showed proteolytic activity when exposed to some of the proteins used in the chemotaxis study. Casein and gelatin were readily hydrolyzed by Bd culture supernatants as evidenced by growth in culture media (1% non-fat dry milk, NFDM) as well as protein separation on zymograms impregnated with these substrates. Elastin, an extracellular matrix protein, was also hydrolyzed by the fungus. Characterization of the elastolytic activity and partial purification of the enzymes responsible was carried out using culture supernatants harvested from 1% NFDM. These characterization studies reveal that Bd secretes proteolytic enzymes that are greatly inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF), a metal ion chelator and a serine protease inhibitor, respectively. This activity was optimal at a pH of 8.0. The divalent metal cations Ca2+ and Mg2+ greatly increased elastolytic activity while Zn2+ was inhibitory. Cross reactivity of a serine protease antiserum from Aspergillus fumigatus was seen with a 110 kDa polypeptide. Immunofluorescence studies also showed this antiserum reacting with Bd zoospores and sporangia. These observations suggest that Bd may produce proteases similar to those produced by other pathogenic fungi that are capable of degrading proteins found in the extracellular matrix. The proteolytic activity exhibited in vitro might aid the organism in its ability to colonize and destroy the epidermis of its amphibian host.