| Summary: | Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, <i>Rhinacanthus nasutus,</i> against the human pathogenic fungus <i>Talaromyces</i><i>marneffei</i>. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The <i>R. nasutus</i> crude protein extract inhibited the growth of <i>T. marneffei</i>. The anti-<i>T. marneffei</i> activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from <i>R. nasutus</i> was partially purified by size fractionation to ≤10, 10–30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti-<i>T. marneffei</i> activity with the MIC and MFC values of 32 to 128 μg/mL and >128 μg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with <i>T. marneffei</i> mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-<i>T. marneffei</i> activity of the 10–30 kDa fraction was abrogated by deletion of <i>gasA</i> and <i>gasC</i>, the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from <i>R. nasutus</i> represent sources for novel drug development.
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