Search

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages’ ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fcγ receptor (FcγR) function, whereby IgG immune complexes cross-link to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

The plasma membrane of eukaryotic cells is commonly believed to contain ordered lipid domains. The interest in understanding the origin of such domains has led to extensive studies on the phase behavior of mixed lipid systems. Three-component phase diagrams, composed of a high melting temperature (Tm) lipid, cholesterol, and a low Tm lipid have been valuable in studying lipid phase behavior. However, developing phase diagrams over the entire composition space and with precise tie-lines requires significant experimental effort. In this study, a machine learning approach was used to predict the Tm of lipids and generate phase diagrams from lipid mixtures. First, artificial neural network (ANN) was used for the prediction of Tm. The network was trained using available Tm data and was able to generate Tm values that closely matched literature results for its testing dataset. This model was then used to predict the Tm for lipids that have not yet been experimentally tested. Then, random forests (RF) and support vector machines (SVM) were trained and tested for their ability to predict a test three-component phase diagram. The model from the RF algorithm was able to generate a diagram that closely matched published results. This model was then used to generate phase diagrams for lipid mixtures at various temperatures and various degrees of unsaturation. This machine learning approach to the generation of lipid phase diagrams has the potential to save significant time and resources in studies of lipid phase behavior.

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.

Antarctic notothenioids are noted for extreme stenothermy, yet underpinnings of their thermal limits are not fully understood. We hypothesized that properties of ventricular membranes could explain previously observed differences among notothenioids in temperature onset of cardiac arrhythmias and persistent asystole. Microsomes were prepared using ventricles from six species of notothenioids, including four species from the hemoglobin-less (Hb−) family Channichthyidae (icefishes), which also differentially express cardiac myoglobin (Mb), and two species from the (Hb+) Nototheniidae. We determined membrane fluidity and structural integrity by quantifying fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and leakage of 5(6)-carboxyfluorescein, respectively, over a temperature range from ambient (0 °C) to 20 °C. Compositions of membrane phospholipids and cholesterol contents were also quantified. Membranes from all four species of icefishes exhibited greater fluidity than membranes from the red-blooded species N. coriiceps. Thermal sensitivity of fluidity did not vary among species. The greatest thermal sensitivity to leakage occurred between 0 and 5 °C for all species, while membranes from the icefish, Chaenocephalus aceratus (Hb−/Mb−) displayed leakage that was nearly 1.5-fold greater than leakage in N. coriiceps (Hb+/Mb+). Contents of phosphatidylethanolamine (PE) were approximately 1.5-fold greater in icefishes than in red-blooded fishes, and phospholipids had a higher degree of unsaturation in icefishes than in Hb + notothenioids. Cholesterol contents were lowest in Champsocephalus gunnari (Hb−/Mb−) and highest in the two Hb+/Mb + species, G. gibberifrons and N. coriiceps. Our results reveal marked differences in membrane properties and indicate a breach in membrane fluidity and structural integrity at a lower temperature in icefishes than in red-blooded notothenioids.

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.

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.

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch highcell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 5 10 3 to 8.08 x 10 3 mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for gamma-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of gamma-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifngamma]. These conditions were considered as suitable conditions for the production of gamma-interferon: 2.5x M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD(600) = 4.5). Under these conditions, dry cell weight of 6 +/- 0.2 g/l and gamma-interferon concentration of 2.15 +/- 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of gamma-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human gamma-interferon. At improved conditions, a dry cell weight of 14 +/- 0.2 g/l, concentration and overall productivity of gamma-interferon 4.2 +/- 0.1 g/l and 420 +/- 10 mg/l h, respectively, were obtained.

In order to increase the productivity of human IL-2 (interleukin-2), a stoichiometric model has been used to determine the most essential amino acids and precise values of their amounts to be added to the culture during expression of human IL-2 (as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2). Experiments were performed to investigate the effect of chosen amino acids and their interactions on expression of human IL-2. Glutamine, a mixture of leucine, aspartic acid and glycine, and a mixture of leucine, glutamine and aspartic acid, were the most effective for the expression of IL-2. The most promising amino acids were then chosen for further experiments at three different levels to determine whether altering their stoichiometry can lead to better expression levels. The optimized value of glutamine in the flask was 0.316 g/l; a mixture of leucine, glutamine and aspartic acid at concentrations of 0.124, 0.316 and 0.212 g/l respectively and of leucine, aspartic acid and glycine in concentrations of 0.124, 0.212, 0.111 g/l respectively were chosen to be added to the flask. The effect of glutamine, as one of the amino acids most influencing the expression of IL-2 in batch and fed-batch high-cell-density cultures, was studied. The results revealed that the amount of expressed IL-2 compared with the control culture increased from 81 to 195 mg/l in the shake flask, 403 to 594 mg/l in the fermentor and 5.15 to 10.01 g/l in the fermentor under fed-batch cultivation.

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids packed in a liquid-ordered state, commonly known as lipid rafts, are believed to exist. While less studied in bacterial cells, reports on the presence of sterol or protein-mediated microdomains in bacterial cell membranes are also appearing with increasing frequency. Recent efforts have been focused on addressing the biophysical and biochemical properties of lipid rafts. However, most studies have been focused on synthetic membranes, mammalian cells, and/or model, non-pathogenic microorganisms. Much less is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and the developing field of microdomains in pathogenic bacteria. The current literature on the structure and function and of microdomains is reviewed and the potential role of microdomains in growth, pathogenesis, and drug resistance of pathogens are discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of the process of pathogenesis and development of raft-mediated approaches for new methods of therapy.