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), apt1 Delta (mutant) and apt1 Delta::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1 Delta 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.
Cryptococcus neoformans, vacuole, flippases, polysaccharide secretion, lipid biosynthesis
NCBI PubMed ID: 29291962Publication DOI: 10.1016/j.bbamcr.2017.12.007Journal NLM ID: 101731731Publisher: Amsterdam: Elsevier
Correspondence: Rodrigues ML
Institutions: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA, Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, Departments of Biochemistry and Molecular Genetics and Microbiology, Stony Brook University, New York, USA, Programa de Pós-Graduação em Química Biológica do Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY, USA, Programa de Pós-Graduação em Biologia Parasitária do Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, USA, Veterans Administration Medical Center, Northport, USA, Division of Infectious Diseases, Stony Brook University, New York, USA, Michael Smith Laboratories, Department of Microbiology and Immunology, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, Canada
Methods: HPLC, extraction, statistical analysis, hydrolysis, sonication, TEM, centrifugation
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