Biology subjects

Arkowitz, R. A.

Publications and source records attributed to Arkowitz, R. A..

3 recordsLinked to original sources

bioRxiv2023

A live-cell ergosterol reporter for visualization of the effects of fluconazole on a human fungal pathogen

Ergosterol, an essential plasma membrane amphipathic lipid, is a major component of the fungal plasma membrane. Most fungal pathogens are sensitive to azole drugs that target ergosterol biosynthesis and resistance/tolerance to azoles is increasingly problematic. Candida albicans is the most prevalent etiology of candidiasis and, in this fungal pathogen, ergosterol rich sub-domains are likely to include sphingolipids, as well as specific membrane proteins, such as multidrug transporters. To investigate the dynamics of ergosterol during the cell cycle and whether drug treatment affects these dynamics in this opportunistic pathogen, we adapted the D4H (domain 4 of the perfringolysin O bacterial toxin) reporter for studying sterol-rich membrane domains. We show that D4H provides a direct readout for the cellular effects of fluconazole and that highly polarized ergosterol is not critical for budding or filamentous growth.

microbiology↗

bioRxiv2022

Two distinct lipid transporters together regulate invasive filamentous growth in the human fungal pathogen Candida albicans

Flippases transport lipids across the membrane bilayer to generate and maintain asymmetry. The human fungal pathogen Candida albicans has 5 flippases, including Drs2, which is critical for filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to the antifungal drug fluconazole and copper ions. We show here that such a flippase mutant also has an altered distribution of phosphatidylinositol 4-phosphate [PI(4)P], and ergosterol. Analyses of additional lipid transporters, i.e. the flippases Dnf1-3, and all the oxysterol binding protein (Osh) family lipid transfer proteins, i.e. Osh2-4 and Osh7, indicate that they are not critical for filamentous growth. However, deletion of Osh4 alone, which exchanges PI(4)P for sterol, in a drs2 mutant can bypass the requirement for this flippase in invasive filamentous growth. In addition, deletion of the lipid phosphatase Sac1, which dephosphorylates PI(4)P, in a drs2 mutant results in a synthetic growth defect, suggesting that Drs2 and Sac1 function in parallel pathways. Together, our results indicate that a balance between the activities of two different classes of lipid transporters regulates invasive filamentous growth, via PI(4)P. In contrast, deletion of OSH4 in drs2 does not restore growth on fluconazole, nor on papuamide A, a toxin that binds PS in the outer leaflet of the plasma membrane, suggesting that Drs2 has additional role(s) in plasma membrane organization, independent of Osh4. As we show that C. albicans Drs2 localizes to different structures, including the Spitzenkorper, we sought to determine if a specific localization of Drs2 is critical for different functions, using a synthetic physical interaction approach to restrict/stabilize Drs2 at the Spitzenkorper. Our results suggest that Drs2 plasma membrane localization is critical for C. albicans growth on fluconazole and papuamide A, but not for invasive filamentous growth.

cell biology↗

bioRxiv2022

Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3{Delta}/{Delta} and ypp1{Delta}/{Delta} mutant was [~]60% and [~]40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4{Delta}/{Delta} mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall {beta}(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3{Delta}/{Delta} had wild-type virulence, the ypp1{Delta}/{Delta} mutant had attenuated virulence and the stt4{Delta}/{Delta} mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1{Delta}/{Delta} mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans. ImportanceWhile the PI-4-kinases Pik1 and Stt4 both produce PI(4)P, the former generates PI(4)P at the Golgi and the latter at the plasma membrane and these two pools are functionally distinct. To address the importance of plasma membrane PI(4)P in Candida albicans, we have generated deletion mutants of the three putative plasma membrane PI-4-kinase complex components and quantified the levels of plasma membrane PI(4)P in each of these strains. Our work reveals that this phosphatidylinositol phosphate is specifically critical for the yeast-to-hyphal transition, cell wall integrity and virulence in a mouse systemic infection model. The significance of this work is in identifying a plasma membrane phospholipid that has an infection specific role, which is attributed to the loss of plasma membrane PI(4)P resulting in {beta}(1,3)-glucan unmasking.

cell biology↗