Analysis of sphingolipids, sterols, and phospholipids in human pathogenic Cryptococcus strains

Abstract

species cause invasive infections in humans. Lipids play an important role in the progression of these infections. Independent studies done by our group and others provide some detail about the functions of these lipids in infections. However, the pathways of biosynthesis and the metabolism of these lipids are not completely understood. To thoroughly understand the physiological role of these lipids, a proper structure and composition analysis of lipids is demanded. In this study, a detailed spectroscopic analysis of lipid extracts from and strains is presented. Sphingolipid profiling by LC-ESI-MS/MS was used to analyze sphingosine, dihydrosphingosine, sphingosine-1-phosphate, dihydrosphingosine-1-phosphate, ceramide, dihydroceramide, glucosylceramide, phytosphingosine, phytosphingosine-1-phosphate, phytoceramide, α-hydroxy phytoceramide, and inositolphosphorylceramide species. A total of 13 sterol species were identified using GC-MS, where ergosterol is the most abundant species. TheP-NMR-based phospholipid analysis identified phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidyl-,-dimethylethanolamine, phosphatidyl--monomethylethanolamine, phosphatidylglycerol, phosphatidic acid, and lysophosphatidylethanolamine. A comparison of lipid profiles among different strains illustrates a marked change in the metabolic flux of these organisms, especially sphingolipid metabolism. These data improve our understanding of the structure, biosynthesis, and metabolism of common lipid groups of and should be useful while studying their functional significance and designing therapeutic interventions.

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

“…Molecular classes detected in this analysis included sphingosines (sphingosine, sphingosine-1-phosphate, phytosphingosine, phytosphingosine-l-phosphate, dihydrosphingosine, dihydrosphingosine-1-phosphate), ceramides (C18, 08:1, C24, C24:l, C26, C26:l, C18-dihy-droceramide, C18:l-dihydroceramide, C24-dihydroceramide, C24:l-dihy-droceramide, C26-dihydroceramide, C26:l-dihydroceramide, dl8:2/ 08:0 h-ceramide, dl8:l/C18:0 h-ceramide, dl9:2/C18:0 h-ceramide), glucosylceramides (dl8:1/08:0 h, dl8:2/C18:0 h, dl9:2/C18:0 h, dl9:2/ C24:0 h, dl9:2/C26:0 h), phytoceramides (08, 08:1, C24, C24:l, C26, C26:l, C18-aOH, C18:l-aOH, C24-aOH, C24:l-aOH, C26-aOH, C26:l-aOH) and inositol phosphorylceramides (IPC-36:0;2, IPC-42:0;2, IPC-44:0;2, IPC-46:0;2, IPC-36:0;3, IPC-36:0;3-X, IPC-36:0;3-Y, IPC-42:0;3, IPC-44:0;3-A, IPC-44:0;3-B, IPC-46:0;3-A, IPC-46:0;3-B, IPC-36:0;4,IPC-42:0;4, IPC-44:0;4, IPC-46:0;4-A, IPC-46:0;4-B, IPC-36:0;5, IPC-42:0;5, IPC-44:0;5, IPC-46:0;5-A, IPC-46:0;5-B), and sterylglucoside. Since the general composition of C. neoformans sphingolipids and sterols has been recently described [43], results presented in this manuscript were normalized to inorganic phosphate content and included only those showing statistically significant differences between WT, mutant and complemented cells.…”

Metabolômica Microbiana: Inovações E Aplicações

“…80 A GC-MS também foi empregada na análise de ácidos graxos submetidos à derivatização 81,82 tanto para avaliações de perfil lipídico 82,83 quanto para identificações específicas. [84][85][86][87] O estudo da produção metabólica em diferentes meios de cultura foi realizado para os micro-organismos Escherichia coli e Saccharomyces cerevisiae onde GC-TOF-MS (do inglês, Gas Chromatography -Time of Flight Mass Spectrometry) foi utilizada na identificação e quantificação dos metabólitos intracelulares. 35 Além da utilização da abordagem alvo-orientada 38 em GC-MS, as abordagens globais também têm sido empregadas em análises de diversos micro--organismos.…”

“…95,96 O modo de monitoramento de reações selecionadas (SRM, do inglês Selected Reaction Monitoring) em LC-MS/MS usando um analisador triplo quadrupolo também tem sido aplicada principalmente na identificação e quantificação de lipídios e outros metabólitos. 84,97,98 As aplicações para LC-MS dentro do contexto da análise metabolômica de micro-organismos são bastante amplas, e a grande quantidade de informação gerada acaba tornando o uso de ferramentas de análises de dados essenciais nesses estudos. 36,64,99,100 Uma abordagem recente que também tem ganhado espaço dentro das análises metabolômicas é a associação da espectrometria de massas com a técnica de mobilidade iônica UHPLC-TWIM-MS (do inglês, Ultra High Performance Liquid Chromatography combined with Traveling Wave Ion Mobility Mass Spectrometry).…”

“…26,37,44,64,67 A PCA foi empregada para avaliar a diferença no perfil lipídico de diferentes espécies de Cryptococcus sp, nos quais os principais compostos responsáveis pela diferenciação foram identificados. 84 Tanto o pré-processamento quanto a aplicação de ferramentas multivariadas foram utilizadas para a classificação de amostras de Bacillus sp. e Brevibacillus sp.…”

Steryl Glycosides in Fungal Pathogenesis: An Understudied Immunomodulatory Adjuvant

“…There are several classes of lipids, including triacyl glycerides, sterols, sphingolipids, phospholipids, glycerophospholipids, and glycolipids. Whether or not certain species of lipids are produced in a cell will vary by the organism, whereas certain lipid species will vary by the kingdom, the genus, or even the strain of an organism [10][11][12]. Indeed, the lipidome, or the totality of all the lipids of a cell, has been an evolving topic of interest for plants, fungi, yeasts, and mammals, including both rodents and humans [13].…”