Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: Potential applications for vaccine development

Cryptococcosis caused by C. neoformans and C. gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Îsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Îsgl1 strain is not CD4+ T-cells dependent. Immunocompromised mice, which lack CD4+ T-cells, are able to control the infection by Îsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Îsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Îsgl1 strain might provide a potential vaccination strategy against cryptococcosis

Quantitative analysis of red blood cell membrane phospholipids and modulation of cell-macrophage interactions using cyclodextrins

© 2020, The Author(s). The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell–cell interactions

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

Abstract Background 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. Methods Infasurf®, a clinically used and commercially available calf lung surfactant extract, was used as lung surfactant model. Infasurf® films were spread on top of an aqueous subphase in a Langmuir trough with smoke particulates from conventional cigarettes or vapor from different flavors of e-cigarettes dispersed in the subphase. Surfactant interfacial properties were measured in real-time upon surface compression while surfactant lateral structure after exposure to smoke or vapor was examined using atomic force microscopy (AFM). Results E-cigarette vapor regardless of the dose and flavoring of the e-liquid did not affect surfactant interfacial properties. In contrast, smoke from conventional cigarettes had a drastic, dose-dependent effect on Infasurf® interfacial properties reducing the maximum surface pressure from 65.1 ± 0.2 mN/m to 46.1 ± 1.3 mN/m at the highest dose. Cigarette smoke and e-cigarette vapor both altered surfactant microstructure resulting in an increase in the area of lipid multilayers. Studies with individual smoke components revealed that tar was the smoke component most disruptive to surfactant function. Conclusions While both e-cigarette vapor and conventional cigarette smoke affect surfactant lateral structure, only cigarette smoke disrupts surfactant interfacial properties. The surfactant inhibitory compound in conventional cigarettes is tar, which is a product of burning and is thus absent in e-cigarette vapor

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

<div><p><i>Cryptococcus neoformans</i> 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 <i>Cryptococcus</i>, primarily utilizing protein-conjugated components of the <i>Cryptococcus</i> polysaccharide capsule as antigen. However, there is currently no vaccine against <i>Cryptococcus</i> in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in <i>C</i>. <i>neoformans</i> 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 <i>C</i>. <i>neoformans</i> infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, <i>Candida utilis</i>, was administered intraperitoneally, prior to infecting mice with a lethal dose of <i>C</i>. <i>neoformans</i>. GlcCer administration prevented the dissemination of <i>C</i>. <i>neoformans</i> 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 <i>C</i>. <i>neoformans</i> infection and can provide a viable vaccination strategy against <i>Cryptococcus</i>.</p></div

Histopathology of lungs and brain of mice infected with <i>C</i>. <i>neoformans</i> and treated with GlcCer vs. untreated mice.

<p>A) Brain of untreated mice stained with H&E. Colonization of <i>C</i>. <i>neoformans</i> (Cn) cells in the brain is shown with arrows. Histology was performed 20 days post-infection. B & C) Brain of infected mice treated with GlcCer or GlcCer + FA. Histology was performed 20 days post-infection. Images showed no abnormality in the brain tissue and no <i>C</i>. <i>neoformans</i> cells was found, confirming CFU data illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153853#pone.0153853.g002" target="_blank"><b>Fig 2C</b>, <b>2D</b> and <b>2G</b></a>) Lungs of untreated mice stained with H&E or mucicarmine. Histology was performed 20 days post-infection. Significant lung inflammation is present (arrowheads in D), where the lung tissue is infiltrated with numerous macrophages, lymphocytes and neutrophils. <i>C</i>. <i>neoformans</i> cells are readily visible in these areas (not shown) and in the alveoli (arrows in G). E & H) Lungs of mice treated with GlcCer and stained with H&E or mucicarmine. Histology was performed 90 days post-infection. Arrows in E illustrate normal lung structure. F & I) Lung of mice treated with GlcCer + adjuvant and stained with H&E or mucicarmine. Histology was performed 90 days post-infection. Arrows in F illustrate normal lung structure. Arrowheads in F illustrate some lung inflammation with no <i>C</i>. <i>neoformans</i> cells present at this particular site of inflammation (I), although they were present in other fields. Three mice per group were used in all histology experiments. The images shown are representative fields of the entire organ. Black bar in A, B an C, 200 μm; black bar in D, E and F, 200 μm; black bar in G, H and I, 40 μm.</p

Detection of GlcCer antibody in the sera of different strains of mice following different route of administration.

<p>A) Sera of CBA/J mice treated with weekly administration of GlcCer + adjuvant using interaperitoneal (IP) or subcutaneous (SC) administration. *, <i>P</i> < 0.05, IgM day 28 or day 56 versus IgM normal serum (day 1). B) Sera of BALB/c mice treated with weekly administeration of GlcCer + FA. Six mice per group were used. Plots show the results of ELISA experiments performed using purified GlcCer as antigen. *, <i>P</i> < 0.05, IgM at day 14, 28, 48, or 56 versus IgM normal serum.</p

GlcCer administration results in partial immunity against cryptococcosis in mouse models of the disease.

<p>(A) Survival of CBA/J mice treated with 20 μg/day of GlcCer or GlcCer + FA and infected intranasally with 5x10⁵ <i>C</i>. <i>neoformans</i> (Cn) cells. The GlcCer and GlcCer + iFA control groups received treatment, but were not infected. The Cn group were infected, but did not receive treatment. Ten mice per group were used. *, <i>P<0</i>.<i>05</i> Cn + GlcCer or Cn + GlcCer + adjuvant versus Cn. (B) Fungal tissue burden in the lungs of the mice treated with 20 μg/day of GlcCer or GlcCer + adjuvant and infected intranasally with 5x10⁵ Cn. Three mice per group per day were used. *, <i>P<0</i>.<i>05</i>, Cn + GlcCer or Cn + GlcCer + adjuvant versus Cn H99 (C). Fungal tissue burden in the brains of the mice treated with 20 μg/day of GlcCer or GlcCer + adjuvant and infected intranasally with 5x10⁵ Cn. Three mice per group per day were used. Day = 0 represents the first day of infection. *, <i>P<0</i>.<i>05</i>, Cn + GlcCer or Cn + GlcCer = adjuvant versus Cn H99.</p

Structure and composition of the analyzed GlcCer’s of <i>Candida utilis</i>.

<p>(A) Structure of GlcCer: i) GlcCer with 4,8-Sphingadienine (d18:2) sphingoid base. ii) GlcCer with 9-Methyl-4,9-Sphingadienine (d19:2) sphingoid base. The additional carbon chain, R = C9 –C27. (B) Composition analysis of GlcCer as analyzed by ESI-MS. The values are Mean ± SEM and n = 3, where ‘n’ represents analysis from 3 independent purifications. Complete list of data points in presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153853#pone.0153853.s004" target="_blank">S1 Table</a></b>.</p