Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

Abstract

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

Keywords: Pythium insidiosum; antifungal drug; evolution; in vitro susceptibility; pythiosis; sterol biosynthetic enzyme.

FIG 1

In vitro drug susceptibility assay of 30 Py. insidiosum isolates against antifungal drugs. The drug concentrations of terbinafine (A) and itraconazole (B) used in the in vitro susceptibility assay are 0.5, 2, 8, 32, 64, and 128 mg/liter. A dot represents the percentage of radial growth of an individual isolate in relation to the growth of the same isolate exposed to no drug (control). *, statistical significance compared to the no-drug control.

FIG 2

Total ion chromatogram of the sterol extracts generated by GC-MS analysis: (A) Standard mixture of sterol end products, (B) sterol extract from the Ca. albicans strain ATCC 90028 (200 mg lyophilized cells), and (C) sterol extract from Py. insidiosum strain Pi-S (1,400 mg lyophilized cells). Peaks 1 to 5 indicate 5α-cholestane (internal control), cholesterol, ergosterol, stigmasterol, and fucosterol, respectively.

FIG 3

Schematic diagram showing enzymes and substrates in the mevalonate (upper part) and sterol biosynthesis (lower part) pathways. ERG1 and ERG11 are the drug targets of terbinafine and itraconazole, respectively.

FIG 4

Phylogenetic analysis of ERG11 orthologs of 17 oomycetes, 3 fungi, 3 algae, and a diatom. A phylogenetic tree was reconstructed based on the neighbor-joining algorithm (see Materials and Methods). The branch support values of at least 70% are shown at corresponding nodes. *, oomycete; #, fungus.