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.     

Growth inhibition of Cryptococcus neoformans by cloned cultured murine macrophages

Cloned and unselected bone marrow-derived macrophage cell lines were obtained from A/J, AKR/J, BIO.A(5R), CBA/J, DBA/2, HPC, NZW, and [NZB X NZW]F₁ mice, and their interactions were studied in vitro with a lightly encapsulated natural serotype A isolate of Cryptococcus neoformans. Growth inhibition of C. neoformans was seen with all of the cell lines, as determined by enumeration of colony-forming units. Inhibition was enhanced by a high concentration (8%) of fresh mouse serum and was the same for serum obtained from AKR/J (C5 deficient) and BIO.A (C5 normal) mice. Macrophage incubation with fresh AKR/J serum which had been absorbed with heat-killed Cryptococcus cells also inhibited C. neoformans growth. Heat-inactivation, EDTA addition or anti-C3 antibody treatment of fresh serum abolished the opsonic activity for C. neoformans, while EGTA addition to fresh serum was without effect on opsonization. In addition, neither IgM nor IgG₁ murine monoclonal antibodies specific for C. neoformans enhanced phagocytosis or killing of the yeast by macrophages. These findings are consistent with the interpretation that C3b is an important modulator of interactions between macrophages and C. neoformans.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The Elastic Basis for the Shape of Borrelia burgdorferi

The mechanisms that determine bacterial shape are in many ways poorly understood. A prime example is the Lyme disease spirochete, Borrelia burgdorferi (B. burgdorferi), which mechanically couples its motility organelles, helical flagella, to its rod-shaped cell body, producing a striking flat-wave morphology. A mathematical model is developed here that accounts for the elastic coupling of the flagella to the cell cylinder and shows that the flat-wave morphology is in fact a natural consequence of the geometrical and material properties of the components. Observations of purified periplasmic flagella show two flagellar conformations. The mathematical model suggests that the larger waveform flagellum is the more relevant for determining the shape of B. burgdorferi. Optical trapping experiments were used to measure directly the mechanical properties of these spirochetes. These results imply relative stiffnesses of the two components, which confirm the predictions of the model and show that the morphology of B. burgdorferi is completely determined by the elastic properties of the flagella and cell body. This approach is applicable to a variety of other structures in which the shape of the composite system is markedly different from that of the individual components, such as coiled-coil domains in proteins and the eukaryotic axoneme.     

The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features

The chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [β-D-Glcp-(1′→4)-β-D-Glcp] linked to the ω-hydroxyl group of α,ω-dihydroxy palmitate [16:0-α,ω-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(α,ω-di-OH)], [18:0-(α,ω-1,ω-tri-OH)], and [18:0-(α,ω-2,ω-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO₃ from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2×10⁻⁵ M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties.     

The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

Background  

Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and C. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. C. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of C. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes.     

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

小檗碱抗新生隐球菌活性和作用机制

【目的】研究小檗碱对新生隐球菌的抗菌活性及其作用机制。【方法】采用微量肉汤稀释法测定小檗碱对新生隐球菌标准菌株和临床分离菌株的最小抑菌浓度,通过棋盘法测定小檗碱与氟康唑、两性霉素B的协同作用,测定小檗碱对隐球菌重要毒力因子的表达,以及对巨噬细胞和隐球菌互作的影响,采用隐球菌感染大蜡螟模型测定小檗碱的体内杀菌活性。【结果】小檗碱是一种杀真菌化合物,在测试的菌株中,最小抑菌浓度(minimum inhibitory concentration, MIC)范围为8-16μg/mL。亚致死剂量小檗碱能够抑制隐球菌荚膜大小、产黑色素能力和有性生殖能力,并能增强巨噬细胞的杀菌能力。锌指转录因子Nrg1介导了上述重要的过程。在隐球菌感染动物模型中,小檗碱能够延长感染大蜡螟的存活时间。【结论】小檗碱在体内外具有优异的抗隐球菌活性,有望作为抗隐球菌药物开发的起始化合物。     

Elastic Properties of Polymer Networks

Many fundamental questions for the understanding of polymer melts and networks are more suitably addressed by current computer simulations than by experiments. The reason is that simulations have simultaneous access to the microscopic structure and the macroscopic behavior of well-defined model systems. The coarse-grained models used often bear little relation to actual chemical species. This is justified by the experimentally established universality of polymer dynamics and no limitation for the test and development of theories which are directed at these universal aspects. The difficulties already encountered on this level will be illustrated for entanglements between polymers which dominate the dynamic in dense systems.For practical purposes it would, of coarse, be desirable to predict the characteristic length and time scales of experimental systems from the chemical structure of the polymer chains. Due to the extremely long relaxation times it is impossible to achieve this in brute-force simulations of truely microscopic models. Systematic coarse-graining combined with a better theoretical understanding seem to offer a practical alternative.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020293     

Properties of acetylcholinesterase from Pisum sativum

Acetylcholinesterase (AChE) from Pisum sativum purified 28 fold showed two closely moving protein bands on polyacrylamide gel electrophoresis, both