The Elastic Properties of the Cryptococcus neoformans Capsule

Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca²⁺ concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties.     

A galactoxylomannan antigen of Cryptococcus neoformans serotype A

The capsular polysaccharide of Cryptococcus neoformans serotype A was fractionated into two chemically and serologically distinct heteroglycans by differential precipitation with cetyltrimethylammonium bromide (CTAB). The major, viscous, acidic glucuronoxylomannan (GXM, 88% w/w) was precipitated with CTAB, while a previously undetected galactoxylomannan (GalXM, 12% w/w) remained in solution. GalXM is characterized by (i) molar ratios of galactose:mannose: xylose:glucuronic acid of 1.9:1.8:1.0.2 and 2% of O-acetyl; (ii) a molecular weight of 275,000 ± 25,000, estimated by gel-permeation chromatography; (iii) extensive degradation by NaIO₄; (iv) precipitation in gel by a lectin, concanavalin A, indicating nonreducing mannosyl termini; and (v) a distinct, immunoprecipitin arc in counterimmunoelectrophoresis. GalXM was further purified by gel-permeation or ion-exchange chromatography.     

Synthesis and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans

The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These structures were derived from N-[5-(1-naphthalenylmethyl)-2-thiazolyl]cyclohexanecarboxamide (41F5), a fungistatic agent previously identified through phenotypic screening (Antimicrob Agents Chemother. 2013;57:4349). Modifications to improve potency and water-solubility of 41F5 focused primarily on the 5-naphthalenyl group, the thiazole core, and the methylene linker between these two structural elements. In general, compounds with lipophilic [5+6] bicyclic ring systems, such as the 7-benzothiophenyl- and 4-indanyl groups, at the 5-position were 2–3 times more active against both fungal species as compared to 41F5. Also, introduction of a carbonyl group at the methylene linker of 41F5 resulted in a 2–3-fold increase in potency. These highly active compounds also showed generally low toxicities against murine P388D1 macrophages resulting in selectivity indices ranging from 63 to >200. Compounds that were highly active against fluconazole-sensitive C. neoformans strains had almost identical activity against fluconazole-resistant variants of this fungus indicating that 14α-demethylase is not their molecular target. Highly active compounds also retained activity against H. capsulatum phagocytosed into P388D1 macrophages.     

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

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.     

Sterol 24-C-methyltransferase: An enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans

Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and ¹H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-²H₃]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.     

Structure and Inhibition of the CO2-Sensing Carbonic Anhydrase Can2 from the Pathogenic Fungus Cryptococcus neoformans

In the pathogenic fungus Cryptococcus neoformans, a CO₂-sensing system is essential for survival in the natural environment (∼ 0.03% CO₂) and mediates the switch to virulent growth in the human host (∼ 5% CO₂). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the “plant-type” β-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors.     

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.     

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.     

Synthesis of angiotensins by cultured granuloma macrophages in murine schistosomiasis mansoni

Components of the angiotensin system are present in granulomas of murine schistosomiasis mansoni. Angiotensins may have immunoregulatory function. Granuloma macrophages cultured for up to 3 days generated substantial angiotensin I (AI) and angiotensin II (AII) which appeared in the culture supernatants. Macrophage monolayers were incubated with 3H-labeled amino acids, and culture supernatants were extracted with acetone and analyzed by HPLC. Radiolabeled products eluted at times corresponding to those of authentic angiotensins. Immunoadsorption of angiotensins with angiotensin antisera removed reputed radiolabeled angiotensins from the supernatants. Treatment of the elution fraction corresponding to that of authentic AI with angiotensin-converting enzyme resulted in the generation of radiolabeled polypeptides which coeluted with authentic AII and His-Leu. Similar experiments conducted with nonadherent granuloma cells devoid of macrophages failed to demonstrate angiotensin production. These results suggest that granuloma macrophages can synthesize angiotensins.     

Structural investigations of two capsular polysaccharides from cryptococcus neoformans

Two capsular polysaccharides from Cryptococcus neoformans serotype A have been shown to be chemically equivalent. One of these polysaccharides was further investigated and shown to consist of a chain of (1→3)-linked d-mannosyl residues, each of which is substituted at O–2 by a d-glucosyluronic acid or d-xylosyl group.     

Growth inhibition of suspension cultured plant cells by ribonucleases

Extracellular, stylar RNases (S-RNases) are produced by self-incompatible, solanaceous plants, such asNicotiana alata, and are thought to be involved in selfpollen rejection by acting selectively as toxins to selfpollen. In this study, the toxicity of RNases to other plant cells was tested by culturing cells ofN. alata andN. plumbaginifolia in the presence ofS-RNases fromN. alata. The growth of cultured cells ofN. plumbaginifolia was inhibited by theS-RNases, but viability was not affected. Growth of cultured cells of oneN. alata selfincompatibility genotype was inhibited by twoS-RNases, indicating that inhibition was not allele specific. Comparisons with the effects of inactivated RNase and other proteins, suggest that the inhibition of growth byS ₂-RNase was partly, but not wholly, due to RNase activity. Heat-denaturedS ₂-RNase was a very effective inhibitor of cell growth, but this inhibitory activity may be a cell surface phenomenon.     

Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide

Treatment of B16-F10 melanoma cells with dimethylsulfoxide (DMSO) or butyric acid (BA) inhibits cell growth and delays tumor appearance in syngeneic mice. Both agents induce morphological changes in these cells. Treatment of melanoma cells with DMSO results in a marked increase in tyrosinase activity and melanin content. BA, on the other hand, does not increase melanin content and decreases tyrosinase activity. The data show that there are marked differences in the effect of DMSO and BA on melanin biosynthesis, whereas both agents inhibit cell growth and cause a delay in tumor appearance. These findings indicate that decreased proliferation of melanoma cells and induction of melanin biosynthesis are not necessarily associated phenomena.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Abstract Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Cloning,characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA

The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3 ± 0.24 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50 °C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.