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Mss4 enrichment to small buds and hyphal filament tips requires the actin cytoskeleton. (A) F-actin is critical for asymmetric distribution of Mss4 in budding cells. A mss4 strain expressing GFP-Mss4 was grown with Dox in the presence or absence of LatA as in Fig. 6 A. Central z-sections and sum projections (10 z-sections) of representative cells from different fields of view are shown (top) and quantification of Mss4 concentration over long axis of cells (bottom) as in Fig. 4 A. (B) F-actin is required for asymmetric Mss4 distribution in germ tubes. A mss4 strain expressing GFP-Mss4 was treated with FCS and then incubated with LatA as in Fig. 6 B. Images as described in Fig. 9 A (top) and quantification of Mss4 concentration over long axis of cells (bottom) as in Fig. 4 A (n = 50 cells). For Fig. 9 (A and B), ni.ex. = 3.
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Membrane lipids have been implicated in many critical cellular processes, yet little is known about the role of asymmetric lipid distribution in cell morphogenesis. The phosphoinositide bis-phosphate PI(4,5)P(2) is essential for polarized growth in a range of organisms. Although an asymmetric distribution of this phospholipid has been observed in s...
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... This plasmid was linearized with NcoI and integrated into the RP10 locus. The Dox-repressible erg11Δ/pTetERG11 and erg25Δ/ pTetERG25 strains were constructed from PY173, a derivative of BWP17 containing the tetracycline-regulatable transactivator TetR-ScHAP4AD, as described (20). The Abp1-GFP and Snf7-GFP strains were generated by homologous recombination, using pFA-GFPγ-URA (21) or pGFP-NAT1 (22) and primer pairs ABP1.P1/ABP1.P2 and SNF71.P1/SNF7.P2 , respectively. ...
Ergosterol, an essential plasma membrane amphipathic lipid, is a major component of the fungal plasma membrane. Most fungal pathogens are susceptible 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.
IMPORTANCE
Ergosterol is a critical membrane lipid in fungi. In Candida albicans , this essential plasma membrane amphipathic lipid is important for interactions with host cells, in particular, host immune responses. Here, we use a live-cell reporter for specifically visualizing ergosterol and show that apical enrichment of this sterol is not critical for budding and filamentous growth in this human fungal pathogen. Our results highlight that this live-cell reporter is likely to be a useful tool in the analyses of azole resistance and tolerance mechanisms, including alterations in drug targets and upregulation of efflux activities.
... The Dox repressible erg11Δ/pTetERG11 and erg25Δ/pTetERG25 strains were constructed from PY173, a derivative of BWP17 containing the tetracycline-regulatable transactivator TetR-ScHAP4AD, as described (20). The Abp1-GFP and Snf7-GFP strains were generated by homologous recombination, using pFA-GFPg-URA (21) or pGFP-NAT1 (22) and primer pairs ABP1.P1/ABP1.P2 and SNF71.P1/SNF7.P2 , respectively. ...
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.
... Proper membrane lipid composition is important for hyphal growth of C. albicans, an important virulence attribute of this pathogenic yeast [34][35][36][37][38][39][40][41]. Since the Ypk1 signaling pathway ...
Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.
... The cytoplasmic membrane lipids are usually arranged as lipid bilayers and the membrane lipids or lipid structures can directly participate in various cellular events as messengers or regulators of signal transduction [9,10]. Some lipids, such as phosphatidylserine [11], Phosphatidylcholine [12], ergosterol [13], Phosphatidylinositol-4-phosphate (PI(4)P) [8], C8-desaturated and C9-methylated glycosylceramide (GlcCer) [14], phosphoinositol diphosphate [15] and ceramide [16], etc., tend to accumulate in specific regions (domains) of the cytoplasmic membrane forming membrane/lipid rafts (MLR) [17], which have evolutionarily conserved roles in the integrity and repair of the plasma membrane. ...
... Similarly, choline, a precursor of membrane lipids, plays an important role in the mycelial morphology of Aspergillus nidulans [18]. In Candida albicans, PI(4,5) phosphoinositol diphosphate (P-2), a cytoplasmic membrane lipid molecule, influences the polarization and host infection ability of the fungi [15]. Ergosterol in Aspergillus fumigatus directly affects its growth and infection ability, and therefore, it is used as a very effective drug target against fungal infection [13]. ...
The cytoplasmic membrane is the fundamental component of all living cells, which participates in various physiological processes, such as material exchange, stress response, cell recognition, signal transduction, cellular immunity, apoptosis, pathogenicity, etc. The normal function of a cytoplasmic membrane requires stable organization of transmembrane protein-lipid microdomains, transmembrane protein-cell wall microdomains, and cytoskeleton-transmembrane protein microdomains. Here, we review the mechanisms and functions of various membrane lipid components, fatty acid content and saturation, membrane curvature, and cell wall and cytoskeleton in plasma membrane homeostasis affecting the pathogenicity of pathogenic fungi. Pathogenic fungi maintains plasma membrane homeostasis and contributes to fungal virulence by maintaining plasma membrane assembly, structural and functional integrity of pathogenic fungi at various stages of cell development through interactions among lipid components of cytoplasmic membranes, transmembrane proteins, cytoskeleton and cell wall components, etc.
... In C. albicans, using the reporter Lactadherin C2 (LactC2) [53][54][55], we observed that the distribution of PS was altered during hyphal growth in the drs2 mutant, as the reporter was visible in an intracellular punctate pattern [36]. To determine the impact of the DRS2 deletion on the distribution of other lipids, shown to be critical for hyphal growth, such as the phosphatidylinositol phosphates PI(4)P and PI(4,5)P 2 [56,57], as well as ergosterol [58], we used specific fluorescent reporters. The distribution of PI(4,5)P 2 appears to be similar in wild-type cells and drs2 filamentous cells (Fig 6A), yet the distribution of PI(4)P was substantially less polarized in the mutant, compared to the wild-type or complemented strains ( Fig 6B). ...
... pExp-pACT1CRIBGFP [48], was used to transform the WT (BWP17) and drs2/drs2 (PY3310) strains. Cdr1-GFP, PH-FAPP1 [E50A,H54A]-GFP, GFP-(PH OSH2[H340R] ) 2 -GFP and GFP-PHPLCδ1-PHPLCδ1-GFP expressing strains were generated as described [56,57]. pDUP5-mScarlet-CtRac1 [91] was used to transform the drs2/DRS2GFP (PY4665) and dnf2/DNF2GFP (PY5746) strains. ...
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 putative 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 Spitzenkörper, we investigated if a specific localization of Drs2 is critical for different functions, using a synthetic physical interaction approach to restrict/stabilize Drs2 at the Spitzenkörper. Our results suggest that the localization of Drs2 at the plasma membrane is critical for C . albicans growth on fluconazole and papuamide A, but not for invasive filamentous growth.
... ; https://doi.org/10.1101/2022.08.30.505893 doi: bioRxiv preprint the drs2 mutant, as the reporter was visible as intracellular punctae [36]. To determine the impact of the DRS2 deletion on the distribution of other lipids, shown to be critical for hyphal growth, such as the phosphatidylinositol phosphates PI(4)P and PI(4,5)P2 [56,57], as well as ergosterol [58], we used specific fluorescent reporters. The distribution of PI(4,5)P2 appears to be similar in wild-type cells and drs2 filamentous cells ( Figure 5A), yet the distribution of PI(4)P was substantially less polarized in the mutant, compared to the wild-type or complemented strains ( Figure 5B). ...
... No reuse allowed without permission. GFP-PHPLCδ1-PHPLCδ1-GFP expressing strains were generated as described [56,57]. ...
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.
... PI 4 P is the major phosphoinositide species in the Golgi apparatus, where it mediates vesicular trafficking of secretory proteins from the Golgi to the plasma membrane and is required for C. albicans to undergo filamentous hyphal growth (3,4). PI 4 P is also found at the plasma membrane (4), where it can be further phosphorylated to PI 4,5 P 2 , which is involved in regulating actin cytoskeleton organization, cell wall integrity, and heat shock response pathways (3,5). The various forms of phosphoinositides are under dynamic regulation in the cell through the action of kinases and phosphatases that are specific for the 39, 49, or 59 position (3). ...
Phosphatidylinositol lipids regulate key processes, including vesicle trafficking and cell polarity. A recent study identified novel roles for phosphatidylinositol 4-phosphate (PI4P) in the plasma membrane of the fungal pathogen Candida albicans, including polarized hyphal growth and cell wall organization. Studies in other organisms were not able to separate the roles of PI4P in the plasma membrane and Golgi, but the C. albicans plasma membrane pool of PI4P could be selectively eliminated by deleting the STT4 kinase, which creates PI4P. Interestingly, stt4Δ mutants were strongly defective in disseminated candidiasis in mice but were not defective in an oral infection. This suggested that abnormal exposure of β-glucan in the mutant cell walls increased recruitment of innate immune cells during disseminated infection, which is not expected to impact oral infection. These results highlight novel roles of PI4P and reinforce the need to test the virulence of C. albicans mutants at different host sites.
... medium containing serum, the cells elongated with protrusions that were roughly one-third the length of wild-type cells after 2 h at 37°C. However, under these repression conditions, the stt4 mutant still expressed STT4 and plasma membrane PI(4)P was still detected (14). We also generated a DAmP (decreased abundance by mRNA perturbation) allele (23) in C. albicans (24), by constructing strains in which one copy of STT4 was deleted and a dominant nourseothricin resistance marker (SAT1) was integrated just 39 of the STT4 stop codon. ...
... In wild-type cells, we observed this green fluorescent protein (GFP)-PH OSH2 -PH OSH2 -GFP reporter localize predominantly at the plasma membrane, yet in efr3, ypp1, and stt4 mutants, there was a decrease in plasma membrane PI(4)P, with a concomitant increase in intracellular Golgi PI(4)P signal (Fig. 5A). Quantification of the reporter fluorescence from these central z-sections, using the Matlab program Hyphal-Polarity (14), indicated that the ratio of mean plasma membrane signal to mean internal signal decreased progressively in the efr3, ypp1, and stt4 mutants, resulting from the decrease in plasma membrane PI(4)P together with the overall increase in internal PI(4)P levels ( Fig. 5B). Closer examination of stt4 mutant cells revealed little to no plasma membrane PI(4)P (Fig. 5A), and this was confirmed by quantification of the normalized fraction of total signal at the plasma membrane, i.e., the plasma membrane signal excluding the Golgi cisternae divided by the total cell signal excluding the Golgi cisternae (Fig. 5C). ...
... These results further confirm that Golgi apparatus and plasma membrane PI(4)P pools are functionally distinct. Our previous analyses of a STT4 repressible strain revealed that upon a 10-fold repression of STT4 transcript, there was a reduction of plasma membrane PI(4,5)P 2 (14). As a result, we examined plasma membrane PI(4,5)P 2 levels in the efr3, ypp1, and stt4 deletion mutants. ...
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 apparatus 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 amounts of plasma membrane PI(4)P in the efr3Δ/Δ and ypp1Δ/Δ mutants were ∼60% and ∼40%, respectively, of that in the wild-type strain, whereas it was nearly undetectable in the stt4Δ/Δ mutant. All three mutants had reduced plas7ma 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 β(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 efr3Δ/Δ mutant had wild-type virulence, the ypp1Δ/Δ mutant had attenuated virulence, and the stt4Δ/Δ mutant caused no lethality. In the mouse model of oropharyngeal candidiasis, only the ypp1Δ/Δ 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. IMPORTANCE While the PI-4-kinases Pik1 and Stt4 both produce PI(4)P, the former generates PI(4)P at the Golgi apparatus 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 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 β(1,3)-glucan unmasking.
... Strikingly, the PI[4,5]P 2 molecular probe had an asymmetric localisation in vegetatively growing hyphal tips, where there was little signal at the hyphal apex, but a strong signal in the sub-apical region. An asymmetric distribution of PI[4,5]P 2 at sites of polar growth has been observed in many organisms including tobacco pollen tubes and in N. crassa, and is required for the yeast to hyphal transition of Candida albicans (Ischebeck et al., 2008;Mähs et al., 2012;Vernay et al., 2012). In contrast to this study, the fluorescent signal for PI[4,5]P 2 in C. albicans, S. cerevisiae and N. crassa was strongest at the hyphal apex (Guillas et al., 2013;Mähs et al., 2012;Vernay et al., 2012). ...
... An asymmetric distribution of PI[4,5]P 2 at sites of polar growth has been observed in many organisms including tobacco pollen tubes and in N. crassa, and is required for the yeast to hyphal transition of Candida albicans (Ischebeck et al., 2008;Mähs et al., 2012;Vernay et al., 2012). In contrast to this study, the fluorescent signal for PI[4,5]P 2 in C. albicans, S. cerevisiae and N. crassa was strongest at the hyphal apex (Guillas et al., 2013;Mähs et al., 2012;Vernay et al., 2012). Interestingly, the subapical concentration of this molecular probe coincided with the so-called endocytic collar, a region with increased endocytic activity (Echauri-Espinosa et al., 2012;Taheri-Talesh et al., 2008). ...
... In contrast, in N. crassa, Mss-4 localized to the membrane only subapical to the hyphal tip and at septa, as well as to non-defined intracellular filamentous structures (Mähs et al., 2012).. while S. cerevisiae Mss4 was found to localize evenly at the PM. This indicates that localisation of Mss4 may not be universally conserved (Audhya and Emr, 2003;Homma et al., 1998;Vernay et al., 2012). Interestingly, the localisation of eGFP-MssD correlated with the localization of the PI[4,5] P 2 molecular probe in mature hyphae of E. festucae, but not at the hyphal tip. ...
Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals.
... Strains were grown in rich medium (yeast extract-peptone-dextrose [YEPD]) at 30°C for all experiments, and induction of filamentous growth was carried out with fetal calf serum (FCS) at 37°C. For doxycycline gene repression, cells were grown in the presence of 20 mg/ml doxycycline (52). Oligonucleotides and synthesized DNA used in this study are listed in Tables S2 and S3. ...
Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In
Candida albicans
