Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67

Abstract

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot SDS. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 non-covalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in extracellular vesicles. The possible role of the presently described structures in fungal-host relationship is discussed.

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

“…In Cryptococcus, PC species, namely, 34 Cryptococcus contain very long chain hydroxylated fatty acyls (24C or 26C). These results are consistent with a previous study on cap67 (an acapsular mutant of Cryptococcus neoformans), which showed similar phosphoglyceride composition (47). Nonetheless, a more targeted study is required to confirm the nonexistence of phosphoglyceride species with hydroxylated fatty acyls.…”

“…This is primarily because the structure and biosynthesis of several fungal lipids is different from mammalian lipids (10). In Cryptococcus, several studies have shown the role of lipids in its growth and virulence, but not many studies have been done to characterize their compositions or the genetic makeup of the biosynthetic pathways (19,20,47,60). In this study, we have determined the compositions of the most common lipids of Cryptococcus, Supplemental Material can be found at: using various spectroscopic approaches.…”

“…Pathways of sphingolipid biosynthesis and genes involved therein are well-characterized in several fungi, such as S. cereviseae, Aspergillus fumigatus, Aspergillus nidulans, C. albicans, Pichia pastoris, and others (34,37,(47)(48)(49)(50)(51)(52)(53)(54). In one example of P. pastoris, the pathway of GlcCer biosynthesis very closely fits with the pathway present in Cryptococcus (19,21,54).…”

“…Cryptococcus have been identified to cause invasive infections in humans, leading to high morbidity (1,5). Among these, C. grubii (H99) and C. gattii strains (R265 and R272) are the most well-studied (1, 5), but little is known about their lipid profiles (19,20,47). In the present study, we have aimed to analyze the major groups of lipids, specifically, sphingolipids, sterols, and phosphoglycerides.…”

Immunoproteomic Analysis Reveals Novel Candidate Antigens for the Diagnosis of Paracoccidioidomycosis Due to Paracoccidioides lutzii

“…The 16 specific immunogenic proteins of P. lutzii can be divided into three main groups. Group 1 comprises seven proteins that have been widely reported in Paracoccidioides under experimental physiological conditions according to STRING analysis, namely, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, citrate synthase, isocitrate lyase, enolase, HSP60-like protein, and an uncharacterized protein (thioredoxin-like superfamily) [ 54 , 70 , 87 , 88 , 89 , 90 , 91 , 92 , 93 ]. Proteins included in group 2 are antigens that already have been described in human PCM, such as triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase [ 70 ], or Paracoccidioides antigens that already have been related to eliciting a humoral immune response in BALB/c mice, such as transketolase, HSP60-like protein, and isocitrate lyase (all shared with PS3) [ 71 ].…”