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 <i>Pythium insidiosum</i> Direct exposure to <i>Py. insidiosum</i> 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 <i>Py. insidiosum</i> However, one patient with an invasive <i>Py. insidiosum</i> infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete <i>Aphanomyces euteiches</i> It remains an open question whether <i>Py. insidiosum</i> is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the <i>in vitro</i> susceptibilities of terbinafine and itraconazole against 30 isolates of <i>Py. insidiosum</i> We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of <i>Py. insidiosum</i> and related oomycetes. The susceptibility assay showed that the growth of each of the <i>Py. insidiosum</i> 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 <i>Py. insidiosum</i> 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 <i>Py. insidiosum</i> In conclusion, <i>Py. insidiosum</i> possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in <i>Py. insidiosum</i> was due to modifications or losses of some of the genes encoding the drug target enzymes.