Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.
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ABSTRACT:
SUBMITTER: Farnoud AM
PROVIDER: S-EPMC4254033 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
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Project description:The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.
| S-EPMC90068 | biostudies-literature
Project description:The crystal structures of the Ca(2+)- and H(+)-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure-function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC(50) for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the K(m) for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E(2) conformation of the ATPase towards the E(1) conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383-2392, 2007) led to a dramatic increase in the apparent stoichiometry of H(+) transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.
| S-EPMC3079066 | biostudies-literature
Project description:In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.
| S-EPMC3045780 | biostudies-literature
Project description:Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP(3)). In Saccharomyces cerevisiae, Plc1-derived IP(3) is a substrate for the inositol polyphosphate kinase Arg82, which converts IP(3) to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP(3) kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP(3) content was reduced in the Cn?plc1 mutant and markedly increased in the Cn?arg1 mutant, while PIP(2) was increased in both mutants. The Cn?plc1 and Cn?arg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (Sc?arg82) strain, the Cn?arg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP(3) causes Ca(2+) release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP(3) as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.
| S-EPMC3639591 | biostudies-literature
Project description:ObjectivesCryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties.MethodsThe C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT-PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [(3)H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [(3)H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest.ResultsIntracellular [(3)H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [(3)H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [(3)H]fluconazole than did controls, and [(3)H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole.ConclusionsWe conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance.
| S-EPMC4398472 | biostudies-literature
Project description:Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. In this study, we find that inositol plays an important role in the transversal of Cryptococcus across the blood-brain barrier (BBB) both in an in vitro human BBB model and in vivo animal models. The inositol stimulation of BBB crossing is dependent upon fungal inositol transporters. The upregulation of genes involved in the inositol catabolism pathway is evident in a microarray analysis. The expression of CPS1, a gene encoding the hyaluronic acid synthase in Cryptococcus, is also upregulated by the inositol treatment. The production of hyaluronic acid increased in cells treated with inositol, which leads to the enhanced binding ability of Cryptococcus cells to the human brain microvascular endothelial cells (HBMECs) constituting the BBB. Overall, our studies provide a mechanism for inositol-dependent Cryptococcus transversal of the BBB, supporting our hypothesis that host inositol utilization by the fungus contributes to Cryptococcus CNS infection.
2012-09-29 | GSE41211 | GEO
Project description:Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.
| S-EPMC2912663 | biostudies-literature
Project description:Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. In this study, we find that inositol plays an important role in the transversal of Cryptococcus across the blood-brain barrier (BBB) both in an in vitro human BBB model and in vivo animal models. The inositol stimulation of BBB crossing is dependent upon fungal inositol transporters. The upregulation of genes involved in the inositol catabolism pathway is evident in a microarray analysis. The expression of CPS1, a gene encoding the hyaluronic acid synthase in Cryptococcus, is also upregulated by the inositol treatment. The production of hyaluronic acid increased in cells treated with inositol, which leads to the enhanced binding ability of Cryptococcus cells to the human brain microvascular endothelial cells (HBMECs) constituting the BBB. Overall, our studies provide a mechanism for inositol-dependent Cryptococcus transversal of the BBB, supporting our hypothesis that host inositol utilization by the fungus contributes to Cryptococcus CNS infection. To understand the effect of inositol on gene expression profiles during cell development, microarray experiments were performed to monitor the genes regulated by inositol. H99 overnight culture was washed with dH2O once, and cells were inoculated on SD medium with or without inositol. Cells were collected from SD plates 24 hr post-inoculation, washed with dH2O, and total RNA was purified. Total RNAs were extracted using Trizol Reagents (Invitrogen) and purified using the Qiagen RNeasy cleanup kit (Qiagen). Cy3 and Cy5-labeled cDNA were generated by incorporating amino-allyl-dUTP during reverse transcription of 5 µg of total RNA as described previously and competitively hybridized to a JEC21 whole-genome array generated previously at Washington University in Saint Louis. After hybridization, arrays were scanned with a GenePix 4000B scanner (Axon Instruments, http://www.axon.com) and analyzed by using GenePix Pro version 4.0 and BRB array tools (developed by Richard Simon and Amy Peng Lam at the National Cancer Institute; http://linus.nci.nih.gov/BRB-ArrayTools.html)
2012-09-29 | E-GEOD-41211 | biostudies-arrayexpress
Project description:Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (Cn?sec14-1 and Cn?sec14-1Cn?sfh5). Similar to the CnPLB1 deletion mutant (Cn?plb1), Cn?sec14-1 was hypovirulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live Cn?sec14-1 and Cn?plb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely related CnSEC14-1 homologue, and CnSFH5, a distantly related SEC fourteen like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of Cn?sec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis.
| S-EPMC3418542 | biostudies-literature
Project description:V-ATPase, which comprises 13-14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.
| S-EPMC10586682 | biostudies-literature