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

Lerksuthirat, Tassanee; Sangcakul, Areeporn; Lohnoo, Tassanee; Yingyong, Wanta; Rujirawat, Thidarat; Krajaejun, Theerapong

doi:10.1128/aac.02352-16

Cited by 61 publications

(78 citation statements)

References 53 publications

(33 reference statements)

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“…This microorganism is unable to synthesize ergosterol because it has an incomplete set of sterol biosynthetic enzymes [2]. This is believed to be the reason behind antifungal treatment failure in pythiosis [1, 3–5].…”

Section: Introductionmentioning

confidence: 99%

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

et al. 2017

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Pythiosis is a severe and life-threatening disease that affects humans and various animal species. We report a model of vascular/disseminated pythiosis occurring after subcutaneous inoculation of 2 x 104 Pythium insidiosum zoospores/mL in immunocompromised BALB/c mice. For this model, we carried out two rounds of experiments. First, we evaluated two protocols of immunosuppression before inoculation: cyclophosphamide at 150 mg/kg (CYP group) and cyclophosphamide 200 mg/kg plus hydrocortisone acetate at 250 mg/kg (CYP+HCA group). It was not possible to obtain mortality in the CYP group; however, the combination of CYP+HCA altered disease outcomes, with mortality rates reaching 60%. Second, we used the CYP+HCA immunosuppression protocol to analyze the histological and immunological statuses triggered by disease. When we inoculated immunocompetent mice with P. insidiosum zoospores, self-healing occurred via increased levels of IL-2, IFN-γ and IL-17A, which are characteristic of the Th1/Th17 cytokine response. For infected and immunosuppressed mice, the cytokine profiles showed high levels of IL-10, IL-6 and TNF-α. Increased IL-10 values are related to fungal infection susceptibility and led us to speculate that infection may be established through suppression of the host immune response. In addition, histopathological evaluation of the kidneys and liver demonstrated the presence of hyphae and the cellular findings suggested an acute vascular inflammation that mimics vascular/disseminated pythiosis in humans. This is the first murine model for pythiosis that is useful both for understanding the pathogenesis of this disease and for evaluating new treatment approaches.

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Section: Introductionmentioning

confidence: 99%

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

et al. 2017

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“…In vitro susceptibility assays. The growths of 27 P. insidiosum strains (Table 1), upon exposure to disulfiram, were measured using the agar-based (12,32) and broth-based (see below) susceptibility methods. The experiments were performed in duplicate.…”

Section: Methodsmentioning

confidence: 99%

“…An agar plug with the organism was placed on the SD agar or immersed in the SD broth containing various concentrations (i.e., 0, 2, 4, 8, 16, 32, 64, and 128 mg/liter) of disulfiram (purity, Ն98%; solvent, dimethyl sulfoxide [DMSO]; manufacturer, Unidrug Innovative Pharma Technologies, Ltd., India) and incubated at 37°C for 2 days. For the agar-based susceptibility method (12,32), two perpendicular diameters of a P. insidiosum colony were averaged, subtracted by the agar plug diameter (5 mm), and divided by half. The actual radial growth of a disulfiram-exposed strain was calculated to the "percent growth" in relation to that of the same strain without drug exposure (control).…”

Section: Methodsmentioning

confidence: 99%

The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum

Krajaejun

Lohnoo

Yingyong

et al. 2019

Antimicrob Agents Chemother

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Pythium insidiosum is an oomycete microorganism that causes a lifethreatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum. Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum. Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum. By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum. The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.

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“…Disease can occur in humans and animals (ie horses, cattle and dogs), and due to its aggressiveness, most cases are treated with extensive surgery to remove the affected organ . Antimicrobial treatment of pythiosis remains controversial once it does not respond appropriately to antifungal drugs, and this resistance was recently linked to the fact that P. insidiosum contains an incomplete set of sterol biosynthetic enzymes …”

Section: Introductionmentioning

confidence: 99%

“…1,2 Antimicrobial treatment of pythiosis remains controversial once it does not respond appropriately to antifungal drugs, and this resistance was recently linked to the fact that P. insidiosum contains an incomplete set of sterol biosynthetic enzymes. 3 Studies on pythiosis have been conducted using rabbits as an experimental model since Witkamp 4 intraperitoneally or intravenously injected hyphae and successfully reproduced pythiosis in rabbits. This model was updated by Miller and Campbell 5 when they implicated the zoospores as the infecting units and demonstrated that immunosuppression is unnecessary for the infection to develop in rabbits.…”

Section: Introductionmentioning

confidence: 99%

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

Verdi

Jesus

Kommers

et al. 2017

Mycoses

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Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

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

Cited by 61 publications

References 53 publications

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

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