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.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop

Structural enzymological studies of trypanosomatid SCP2-thiolase reveal its unique reaction mechanism. These studies suggest that this enzyme is a biosynthetic enzyme, possibly involved in the sterol biosynthetic pathway, which is essential in the human pathogenic stages of some trypanosomatid parasites.

Possible Inhibitory Molecular Mechanism of Farnesol on the Development of Fluconazole Resistance in Candida albicans Biofilm

ABSTRACTCandida albicansbiofilm infections are usually treated with azole antifungals such as fluconazole. However, the development of resistance to this drug inC. albicansbiofilms is very common, especially in immunocompromised individuals. The upregulation of the sterol biosynthetic pathway geneERGand the efflux pump genesCDRandMDRmay contribute to this azole tolerance inCandidaspecies. We hypothesize that farnesol, an endogenous quorum sensing molecule with possible antimicrobial properties which is also the precursor of ergosterols inC. albicans, may interfere with the development of fluconazole resistance inC. albicansbiofilms. To test this hypothesis, MICs were compared and morphology changes were observed by confocal laser scanning microscopy (CLSM) for farnesol-treated and -untreated and fluconazole-resistant groups. The expression of possible target genes (ERG11,ERG25,ERG6,ERG5,ERG3,ERG1,MDR1,CDR1, andCDR2) in biofilms was analyzed by reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR) to investigate the molecular mechanisms of the inhibitory effects of farnesol. The results showed a decreased MIC of fluconazole and thinner biofilms for the farnesol-treated group, indicating that farnesol inhibited the development of fluconazole resistance. The sterol biosynthetic pathway may contribute to the inhibitory effects of farnesol, as the transcription levels of theERG11,ERG25,ERG6,ERG3, andERG1genes decreased in the farnesol-treated group.