Pyrifenox, an ergosterol inhibitor, differentially affects Cryptococcus neoformans and Cryptococcus gattii

Silva, Vanessa K A; May, Robin C.; Rodrigues, Márcio L.

doi:10.1093/mmy/myz132

Cited by 4 publications

(4 citation statements)

References 40 publications

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“…AMB, an antifungal that directly disrupts the plasma membrane through sequestration of ergosterol (46), was significantly more potent with increases in the pH of the growth environment. Similarly, fluconazole and pyrifenox, drugs that inhibit the ergosterol biosynthesis pathway (47,69), were also more effective at alkaline pH. These results reflect similar findings in Aspergillus species treated with itraconazole and AMB (48).…”

Section: Discussionsupporting

confidence: 77%

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

Brown

Telzrow

Saelens

et al. 2020

mBio

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The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various microenvironments encountered during a host infection. For example, the fungus-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans. However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis. IMPORTANCE The work described here further elucidates how microorganisms sense and adapt to changes in their environment to establish infections in the human host. Specifically, we uncover a novel mechanism by which an opportunistic human fungal pathogen, Cryptococcus neoformans, responds to increases in extracellular pH in order to survive and thrive within the relatively alkaline environment of the human lung. This mechanism, which is intimately linked with fungal membrane sterol homeostasis, is independent of the previously well-studied alkaline response Rim pathway. Furthermore, this ergosterol-dependent alkaline pH response is present in Candida albicans, indicating that this mechanism spans diverse fungal species. These results are also relevant for novel antimicrobial drug development as we show that currently used ergosterol-targeting antifungals are more active in alkaline environments.

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Section: Discussionsupporting

confidence: 77%

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

Brown

Telzrow

Saelens

et al. 2020

mBio

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“…AMB, an antifungal that directly disrupts the plasma membrane through sequestration of ergosterol (41), was significantly more potent with increases in the pH of the growth environment. Similarly, fluconazole and pyrifenox, drugs that inhibit the ergosterol biosynthesis pathway (42, 64), were also more effective at alkaline pH. These results reflect similar findings in Aspergillus species treated with itraconazole and AMB (43).…”

Section: Resultssupporting

confidence: 73%

“…The significant increase in AMB activity against this mutant strain with reduced ergosterol content is consistent with our model that disruption in fungal sterols leads to pH-sensitivity. Furthermore, in a drug disc diffusion assay using Pyrifenox, a drug used to treat phytopathogens through inhibition of ergosterol biosynthesis (42), there was a significantly greater zone of clearance and inhibition of growth of wildtype C. neoformans cells when grown on media buffered to pH 8 compared to pH 5.5 (Fig 6A).…”

Section: Resultsmentioning

confidence: 99%

Sterol-response pathways mediate alkaline survival in diverse fungi

Brown

Telzrow

Saelens³

et al. 2020

Preprint

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33The ability for cells to maintain homeostasis in the presence of extracellular stress is 34 essential for their survival. Stress adaptations are especially important for microbial pathogens 35 to respond to rapidly changing conditions, such as those encountered during the transition from 36 the environment to the infected host. Many fungal pathogens have acquired the ability to quickly 37 adapt to changes in extracellular pH to promote their survival in the various micro-environments 38 encountered during a host infection. For example, the fungal-specific Rim/Pal alkaline response 39 pathway has been well characterized in many fungal pathogens, including Cryptococcus 40 neoformans. However, alternative mechanisms for sensing and responding to host pH have yet 41 to be extensively studied. Recent observations from a genetic screen suggest that the C. 42neoformans sterol homeostasis pathway is required for growth at elevated pH. This work 43 explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, 44and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for 45 growth in an alkaline environment, and that an elevated pH is sufficient to induce Sre1 46 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the 47 biosynthesis of ergosterol, but is not dependent on Rim pathway signaling, suggesting that 48 these two pathways are responding to alkaline pH independently. Furthermore, we discover that 49 C. neoformans is more susceptible to membrane-targeting antifungals in alkaline conditions 50 highlighting the impact of micro-environmental pH on the treatment of invasive fungal infections. 51Together, these findings further connect membrane integrity and composition with the fungal pH 52 response and pathogenesis. 53Diverse cell types, from simple unicellular microorganisms to complex multicellular 55 eukaryotes, interpret alterations in extracellular pH as a common signal for changes in the 56 external environment. Pathogenic microorganisms are often uniquely exposed to wide 57 fluctuations in pH as they move between various micro-environments in the human host. Among 58 these, fungi that cause invasive fungal infections (IFIs) have acquired the ability to rapidly adapt 59 to changes in extracellular pH to promote their survival during an infection. The shift of a fungal 60 pathogen from an acidic external environment to the neutral/alkaline pH of the mammalian host 61 is associated with the activation of the fungal-specific Rim/Pal signaling pathway, triggering 62 cellular changes important for survival in these new conditions. These changes include 63 alterations in the cell wall, often accompanied by larger morphological transitions that promote 64 host colonization. In the common fungal pathogen Candida albicans, pH-directed cellular 65 responses includes the ability to transition between yeast-like growth and invasive hyphal forms 66 (1-3). The opportunistic human fungal pathogen and basidiomycete yeast Cryptococcus 67 ne...

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“…The cytotoxicity of fenbendazole to macrophages was determined using the Cytotox 96 ® non-radioactive cytotoxicity assay kit (Promega, catalog number G1780), following the manufacturer’s recommendations. As recently described by our group (43), macrophages were used for cell viability assays due to their fundamental roles in the control and/or dissemination of cryptococci (27). RAW 264.7 macrophages (10 5 cells/well in DMEM medium supplemented with 10% FBS were treated with 0.006 to 5.98 μg/ml fenbenzaole, alone or in combination with 0.06 μg/ml amphotericin B for 24 h at 37 °C in DMEM supplemented with 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

Fenbendazole controlsin vitrogrowth, virulence potential and animal infection in theCryptococcusmodel

Oliveira

Joffe

Simon

et al. 2020

Preprint

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AbstractThe human diseases caused by the fungal pathogens Cryptococcus neoformans and C. gattii are associated with high indices of mortality, and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anti-cryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anti-cryptococcal benzimidazole. Fenbendazole was inhibitory against 30 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anti-cryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anti-cryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mice model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

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Pyrifenox, an ergosterol inhibitor, differentially affects Cryptococcus neoformans and Cryptococcus gattii

Cited by 4 publications

References 40 publications

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

Sterol-response pathways mediate alkaline survival in diverse fungi

Fenbendazole controlsin vitrogrowth, virulence potential and animal infection in theCryptococcusmodel

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