Identification and partial characterization of an elastolytic protease in the amphibian pathogen Batrachochytrium dendrobatidis

Abstract

Batrachochytrium dendrobatidis (Bd) is a fungus that causes chytridiomycosis, a disease that has been implicated as a cause of amphibian population declines worldwide. Infected animals experience hyperkeratosis and sloughing of the epidermis due to penetration of the keratinized tissues by the fungus. These symptoms have led us to postulate that Bd produces proteases that play a role in the infection process. Here, we show that Bd is capable of degrading elastin in vitro, a protein found in the extracellular matrix of the host animal. Elastolytic enzyme activity was partially purified using ion exchange chromatography and size-exclusion filtration from cultures grown in inducing media. The elastolytic activity of the purified fraction had a pH optimum of 8, was strongly inhibited by EDTA and phenylmethylsulfonyl fluoride (PMSF), and was partially inhibited by an elastasespecific inhibitor. This activity was also enhanced by the presence of Mg 2+ and Ca 2+ but not Zn 2+. An antiserum directed against Aspergillus fumigatus serine protease (Alp) was found to react with a polypeptide of approximately 110 kDa from the purified material. Using immunofluorescence, this antiserum was also observed to react with zoospores and sporangia grown on toad skin. 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.

Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytrium dendrobatidis in Panama

“…Batrachochytrium dendrobatidis produces an assortment of proteolytic enzymes (Symonds et al 2008, Moss et al 2010. Though there is, as yet, no evidence among reptiles of the lethal, epizootic chytridiomycosis that has afflicted amphibian populations in many parts of the world, it does not follow that B. dendrobatidis infection in reptiles, should it occur, is necessarily benign.…”

“…Chytridiomycosis is fatal to amphibians and is considered to be exclusively an amphibian disease (Berger et al 1998, Wake 2007. Although it had previously been thought that B. dendrobatidis likely feeds on keratin in amphibian skin (Berger et al 1998, Green & Kagarise Sherman 2001, recent work has shown this to be unlikely (Voyles et al 2011) and that proteases secreted by B. dendrobatidis apparently degrade proteins such as elastin in the extracellular matrix of skin tissue (Moss et al 2010).…”

Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection

“…Consequently, as Berger et al (28) hypothesized in their seminal study discovering B. dendrobatidis, we postulated that B. dendrobatidis might be producing a factor that can cause pathology in the absence of actual infection; for instance, B. dendrobatidis produces proteolytic enzymes known to degrade host tissues (22,(29)(30)(31). To test this hypothesis, we exposed P. alleni to (i) an unfiltered B. dendrobatidis + inoculum, (ii) a B. dendrobatidis + inoculum where all of the zoospores and zoosporangia were removed with a 0.7 μm filter, or (iii) a control B. dendrobatidis − inoculum filtered through a 0.7 μm filter (n = 5 per treatment).…”

“…35 and 36). Whether these pathology-inducing chemicals released by B. dendrobatidis are known proteases (22,(29)(30)(31) and are the cause of B. dendrobatidis-induced electrolyte imbalance and cardiac arrest in amphibians (37) remains to be tested.…”

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

“…In analogy with other pathogenic fungi e.g. Candida albicans [17] and dermatophytes [18], most probably a range of digestive enzymes capable of degrading skin components enable penetration of Bd into the host cells [16], [19], [20].…”