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

Studies on MembraneProteins of Mammalian Gamete Cell with Special Reference to a Heparin Binding Protein.

Reproduction is a device that is evolved for the survival of diverge species of organism by producing continues streams of new generation. This becomes imperative in order to escape extinction of the species since, no organism is immortal. Reproduction in mammals requires the existence of two separate sexes; male and female, each sex produces specialized sex cells known as the gametes in their primary sex organs, the gonads. Testes are the male gonad and the spermatozoon form the mature male gamete, while ovary is the female gonad and the mature female gamete are called the ova. The process involved in the union of a spermatozoon with an ovum is fertilization which gives rise to a new individual. This process in human being has raised concern over the population bloom on one hand and infertility on the other hand. Infertility is not a curse as many societies still considers, but rather, it is a biological disorder and basic research is most important to address both the above concern

The N-Terminal of a Heparin-Binding Sperm Membrane Mitogen Possess Lectin-like Sequence

Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids—1A D T I V A V E L D T Y P N14—correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A

Protein Tyrosine Phosphorylation of a Heparin-Binding Sperm Membrane M(HBSM) is Associated with Capacitation and Acrosome Reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase 32Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-L-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reactio

Hydroxyurea treatment inhibits proliferation of Cryptococcus neoformans in mice

The fungal pathogen Cryptococcus neoformans (Cn) is a serious threat to immunocompromised individuals, especially for HIV patients who develop meningoencephalitis. For effective cryptococcal treatment, novel antifungal drugs or innovative combination therapies are needed. Recently, sphingolipids have emerged as important bioactive molecules in the regulation of microbial pathogenesis. Previously we reported that the sphingolipid pathway gene, ISC1, which is responsible for ceramide production, is a major virulence factor in Cn infection. Here we report our studies of the role of ISC1 during genotoxic stress induced by the antineoplastic hydroxyurea (HU) and methylmethane sulfonate (MMS), which affect DNA replication and genome integrity. We observed that Cn cells lacking ISC1 are highly sensitive to HU and MMS in a rich culture medium. HU affected cell division of Cn cells lacking the ISC1 gene, resulting in cell clusters. Cn ISC1, when expressed in a Saccharomyces cerevisiae (Sc) strain lacking its own ISC1 gene, restored HU resistance. In macrophage-like cells, although HU affected the proliferation of WT Cn cells by 50% at the concentration tested, HU completely inhibited Cn isc1-delta cell proliferation. Interestingly, our preliminary data show that mice infected with WT or Cn isc1-delta cells and subsequently treated with HU had longer lifespans than untreated, infected control mice. Our work suggests that the sphingolipid pathway gene, ISC1, is a likely target for combination therapy with traditional drugs such as HU

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes

Abstract Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug’s TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3’-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription

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

Detection of cytokines in the medium of mice liver mononuclear cells following glycolipid treatment.

<p>Detection of A) IL-4, B) IFN-ϒ. C) TNF-α, D) IL-6, E) IL-12p40, and F) IL-1β. Plots are the result of ELISA experiments performed on the media of cells after 48 hours of incubation with various concentration of β-GlcCer or 100 ng/ml of α-GalCer. * <i>P < 0</i>.<i>05</i>, GalCer versus medium, DMSO or GlcCer groups.</p

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

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