Antifungal Activity and Molecular Mechanisms of Partial Purified Antifungal Proteins from Rhinacanthus nasutus against Talaromyces marneffei.

Juthatip Jeenkeawpieam,Sittiruk Roytrakul,Monsicha Pongpom,Alex Andrianopoulos,Supachai Yodkeeree

doi:10.3390/jof6040333

Abstract

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, Rhinacanthus nasutus, against the human pathogenic fungus Talaromyces marneffei. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei. The anti-T. marneffei 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 R. nasutus 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-T. marneffei 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 T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-T. marneffei activity of the 10–30 kDa fraction was abrogated by deletion of gasA and gasC, 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 R. nasutus represent sources for novel drug development.

Highlights

Talaromyces marneffei causes a life-threatening mycosis, primarily affecting immunocompromised residents and travellers in Southeast Asia, southern China, and northeasternIndia [1,2]
At 37 ◦ C, T. marneffei grows in a unicellular yeast form that represents the pathogenic form in human infections
Rhinacanthus nasutus is a well-known, versatile plant that has been used for treatment of various infectious diseases and cancers in Thai alternative medicine for many years [17]

Summary

Introduction

Talaromyces marneffei causes a life-threatening mycosis (talaromycosis), primarily affecting immunocompromised residents and travellers in Southeast Asia, southern China, and northeasternIndia [1,2]. Amphotericin B and itraconazole are effective drugs in the treatment of talaromycosis [3,4,5]. Other azoles such as voriconazole are effective, whereas echinocandins such as anidulafungin showed only moderate activities against the pathogenic yeast phase of T. marneffei [6,7,8]. Resistance to some antifungal drugs has been reported for T. marneffei, especially for fluconazole [3,4,5,6,7,8]. An increasing number of T. marneffei infections have been reported among non-HIV-infected.

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