TY - JOUR
T1 - The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans
AU - Rizzo, Juliana
AU - Colombo, Ana C.
AU - Zamith-Miranda, Daniel
AU - Silva, Vanessa K.A.
AU - Allegood, Jeremy C.
AU - Casadevall, Arturo
AU - Del Poeta, Maurizio
AU - Nosanchuk, Joshua D.
AU - Kronstad, James W.
AU - Rodrigues, Marcio L.
N1 - Funding Information:
This work was supported by grants from the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq): Grants 443586/2014-4 and 300699/2013-1 and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ): E-26/202.997/2015 . The authors also acknowledge support from the Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças de Populações Negligenciadas (INCT-IDPN). J.R. was a Ph.D. student affiliated to Programa de Pós-Graduação em Química Biológica, IBqM/UFRJ and was partially supported by an Interhemispheric Research Training Grant in Infectious Diseases, Fogarty International Center D43TW007129 at the Nosanchuk Laboratory (Albert Einstein College of Medicine, NY). We are especially grateful to Frank Macaluso, Leslie Cummings, and Geoff Perumal at the Einstein Analytical Imaging Facility for assistance with electron microscopy sample preparation and imaging. We would like to thank Robert Rieger at the Stony Brook Proteomics Center for analysis of sphingolipids and sterylglucoside with mass spectrometry. We also acknowledge the contribution of the VCU Lipidomics/Metabolomics Core to phospholipid analysis, supported by the NIH-NCI Cancer Center Support Grant P30 CA016059 to the VCU Massey Cancer Center, as well as a shared resource grant (S10RR031535) from the National Institutes of Health. JDN is partially supported by NIH AI52733 AI1033142, and AI124797. AC is supported in part by AI033142 24, AI052733 and HL059842 17. JWK is supported by grant MOP-13234 from the Canadian Institutes of Health Research and NIH grant AI053721. MDP is supported by NIH grants AI116420, AI125770, and by a Merit Review grant I01BX002624 from the Veterans Affairs Program in Biomedical Laboratory Research and Development.
Publisher Copyright:
© 2017 Elsevier B.V.
PY - 2018/3
Y1 - 2018/3
N2 - Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.
AB - Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.
KW - Cryptococcus neoformans
KW - Flippases
KW - Lipid biosynthesis
KW - Polysaccharide secretion
KW - Vacuole
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U2 - 10.1016/j.bbamcr.2017.12.007
DO - 10.1016/j.bbamcr.2017.12.007
M3 - Article
C2 - 29291962
AN - SCOPUS:85041624807
SN - 0167-4889
VL - 1865
SP - 532
EP - 541
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
IS - 3
ER -