Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of <em>Cryptococcus neoformans</em>

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

Stony Brook University

Ohio University

Perfluoroalkyl acids (PFAAs) are persistent environmental contaminants resistant to biological and chemical degradation due to the presence of carbon-fluorine bonds. These compounds exhibit developmental toxicity in vitro and in vivo. The mechanisms of toxicity may involve partitioning into lipid bilayers. We investigated the interaction between perfluorobutane sulfonate (PFBS), an emerging PFAA, and model phosphatidylcholine (PC) lipid assemblies (i.e., dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholine) using fluorescence anisotropy and Langmuir monolayer techniques. PFBS decreased the transition temperature and transition width of PC bilayers. The apparent membrane partition coefficients ranged from 4.9 × 10(2) to 8.2 × 10(2). The effects on each PC were comparable. The limiting molecular area of PC monolayers increased, and the surface pressure at collapse decreased in a concentration-dependent manner. The compressibility of all three PCs was decreased by PFBS. In summary, PFBS disrupted different model lipid assemblies, indicating potential for PFBS to be a human toxicant. However, the effects of PFBS are not as pronounced as those seen with longer chain PFAAs.

Disruption of phosphatidylcholine monolayers and bilayers by perfluorobutane sulfonate.

Recent interest in using submicrometer particles for industrial and therapeutic purposes has led to concerns about their interactions with biological membranes. The mechanisms of particle-membrane interactions are not well understood resulting in contradictory reports on the effects of particles on membrane interfacial properties. In this study, the interactions between negatively charged polystyrene particles (200 nm) and monolayers of dipalmitoyl phosphatidylcholine (DPPC) were investigated. Surface pressure, surface potential, and surfactant microstructure studies were conducted to monitor the interfacial properties of DPPC monolayers spread on a subphase in which particles were dispersed. At a concentration of 0.1 g/L, particles caused a partial collapse of the monolayer. DPPC monolayers spread on a particle-laden subphase also exhibited higher surface potential and increased ratio of ordered domains supporting the presence of a more compact monolayer. These results suggest that particles penetrated the air-water interface thereby altering monolayer packing at the interface. These findings are contrary to our previous work where particles injected into the subphase beneath a DPPC monolayer did not penetrate the interface confirming that the sequence of particle and monolayer addition can alter particle-monolayer interactions. These studies may partially explain the varying results reported in previous studies.

Interaction of dipalmitoyl phosphatidylcholine monolayers with a particle-laden subphase.

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies.

Plasma membrane lipids and their role in fungal virulence.

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known. In this study, we used genetic engineering to alter the chemical structure of fungal GlcCer and studied its effect on fungal growth and pathogenicity. Here we show that unsaturation in C8 and methylation in C9 is required for virulence in the mouse model without affecting fungal growth in vitro or common virulence factors. However, changes in GlcCer structure led to a dramatic susceptibility to membrane stressors resulting in increased cell membrane permeability and rendering the fungal mutant unable to grow within host macrophages. Biophysical studies using synthetic vesicles containing GlcCer revealed that the saturated and unmethylated sphingolipid formed vesicles with higher lipid order that were more likely to phase separate into ordered domains. Taken together, these studies show for the first time that a specific structure of GlcCer is a major regulator of membrane permeability required for fungal pathogenicity.

Changes in glucosylceramide structure affect virulence and membrane biophysical properties of Cryptococcus neoformans.

Despite their growing popularity, the potential respiratory toxicity of electronic cigarettes (e-cigarettes) remains largely unknown. One potential aspect of e-cigarette toxicity is the effect of e-cigarette vapor on lung surfactant function. Lung surfactant is a mixture of lipids and proteins that lines the alveolar region. The surfactant layer reduces the surface tension of the alveolar fluid, thereby playing a crucial role in lung stability. Due to their small size, particulates in e-cigarette vapor can penetrate the deep lungs and come into contact with the lung surfactant. The current study sought to examine the potential adverse effects of e-cigarette vapor and conventional cigarette smoke on lung surfactant interfacial properties.

Electronic cigarette vapor alters the lateral structure but not tensiometric properties of calf lung surfactant.

Broad interests reap benefits for science.

Insulin fibrillation is caused by the interaction of partially unfolded protein molecules resulting in the formation of insoluble aggregates. Engineered nano- and submicron particles have been reported to accelerate or inhibit the fibrillation of insulin. However, the mechanism by which engineered particles regulate insulin fibrillation kinetics is not fully understood. In the current study, insulin fibrillation in the presence of surface engineered, amine- or sulfate-modified polystyrene particles (200 nm) was investigated. Both particles were found to enhance the rate of protein fibrillation at high particle concentrations (1:1 mass ratio of proteins to particles) as evidenced by increased fluorescence of thioflavin T. A significant amount of proteins was found to adsorb on the surface of both particles, suggesting that the particles served as a nucleation site for fibrillation. Insulin fibrillation was associated with a change in the protein's secondary structure from α-helix to β-sheet. This change of structure was not significantly affected by the presence of particles. Monte Carlo simulations of protein fibrillation in the presence of different surfaces confirmed experimental results and demonstrated that protein fibrillation is enhanced as the affinity of protein molecules toward the surface increases. Along with increasing the local concentration of proteins, the presence of a surface stabilizes unfolded intermediates, which are precursors to fibrillation. These results provide insight into the mechanisms by which engineered particles promote protein fibrillation and can be important in the context of nanotoxicity.

Submicron polymeric particles accelerate insulin fibrillation by surface adsorption.

 is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a  mutant lacking the gene encoding sterylglucosidase (Δ) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δ strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the  gene in the acapsular mutant Δ to generate a double mutant (strain Δ/Δ) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δ/Δ mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of  against  infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δ EVs as a vaccination strategy for cryptococcosis. The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

 Glucuronoxylomannan and Sterylglucoside Are Required for Host Protection in an Animal Vaccination Model.

Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer.

Amir M. Farnoud

Department of Molecular Genetics and Microbiology

Stony Brook University

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Amir M. Farnoud

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Genetics and Microbiology

Stony Brook University

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Department of Molecular Genetics and Microbiology