Infectivity of amphibian chytrid fungi requires metalloprotease-driven spore development and germ tube formation

Chytridiomycosis, caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), drives global amphibian declines. Infection of a susceptible host begins when zoospores attach to the skin and extend germ tubes to penetrate the epithelial tissue. Proteases, particularly metalloproteases, are thought to degrade extracellular matrix components, facilitating tissue invasion, but their roles in spore development and early host colonization remain unclear. We demonstrated that metalloproteases are rapidly secreted by zoospores and are critical for the transition from attachment to germ tube outgrowth and subsequent spore development, important steps in infectivity. Two protease inhibitor mixtures, PI 8340 and PI 8215, were tested in vitro, with PI 8215 containing the metalloprotease-specific inhibitor 1,10-Phenanthroline. PI 8340 had minimal effects on zoospore viability at concentrations up to 0.33% (v/v) for Bd and 1% (v/v) for Bsal. In contrast, PI 8215 impaired spore viability in a dose-dependent manner, with significant effects from 0.0025% (v/v, 12.5 µM 1,10-Phenanthroline) in Bd and 0.005% (v/v, 25 µM 1,10-Phenanthroline) in Bsal. Sublethal concentrations of PI 8215 (0.005% v/v) and 1,10-Phenanthroline (25–50 µM) suppressed germ tube formation, which resumed once the inhibitors were removed. Protease activity assays confirmed that metalloproteases are secreted during early spore stages, with activity detectable as early as 2 h after spore collection, highlighting their role in initial spore development. Metalloprotease activity depends on specific metal cofactors, as growth inhibition by 1,10-Phenanthroline could be partially rescued in Bsal with Zn²⁺ (100 µM) or Mg²⁺ (20 mM). In Bd, recovery was limited and largely independent of cation supplementation. Ex vivo infection of Pleurodeles waltl skin confirmed the in vitro findings, as a lethal concentration (100 µM) significantly reduced Bsal loads after five days, whereas a sublethal concentration (20 µM) had no detectable effect, possibly due to protective cations in host tissue. Metalloproteases are critical for spore development. Inhibition of these enzymes stalls germ tube formation and spore development, highlighting their central role in the earliest stages of chytrid development and suggesting their importance for infectivity.