c neoformans serotype a strains h99  (ATCC)

Journal: Carbohydrate polymers

Article Title: Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

doi: 10.1016/j.carbpol.2022.119547

Figure Lengend Snippet: Effects of lyophilization on NMR signals of C. neoformans EPS. One-dimensional 1 H solution NMR spectra and insets expanded vertically by factors of 10 at 60 °C for a native (A) preparation compared with preparations which were lyophilized and solvated with water at time 0 (B) and after 14 days (C); the three spectra are overlayed in (D). SRG region peaks which were integrated indicated as I, II, and III as the motif they belong to is unknown. Peak integrals for the SRG region of the solution-state spectra were compared by setting the respective DSS signals to 1.0. One-dimensional 1 H solid-state NMR (ssNMR) spectra obtained at room temperature with 15-kHz magic-angle spinning are shown for native (E: concentrated, partially dehydrated) and lyophilized (F: partially rehydrated) samples, normalized according to sample mass. The chemical shifts of the solution- and solid-state spectra were referenced to DSS at 0.0 ppm and water at 4.8 ppm, respectively. The sharp peaks in the ssNMR at 2.9 and 4.8 ppm are attributed to glycine and water, respectively.

Article Snippet: C. neoformans serotype A strain H99 (ATCC 208821) cells were inoculated in Sabouraud rich medium from a frozen stock and grown for two days at 30 °C with agitation (150 rpm).

Techniques: Lyophilization

Journal: Carbohydrate polymers

Article Title: Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

doi: 10.1016/j.carbpol.2022.119547

Figure Lengend Snippet: Effects of lyophilization on solid-state 13 C NMR spectra of EPS. 150 MHz 13 C NMR spectra of C. neoformans EPS samples obtained with 15 kHz magic-angle spinning (MAS), comparing samples that were native (partially dehydrated) (left) and lyophilized (partially rehydrated) (right). DPMAS experiments with short (2 s) delays between successive cycles of signal acquisition, favoring carbon moieties that tumble rapidly in many directions. Sharp resonances at 40 and 170 ppm are attributed to glycine in the culture media. (No EPS signals are observed in CPMAS experiments that favor rigid carbon moieties with nearby hydrogens.)

Article Snippet: C. neoformans serotype A strain H99 (ATCC 208821) cells were inoculated in Sabouraud rich medium from a frozen stock and grown for two days at 30 °C with agitation (150 rpm).

Techniques: Lyophilization

Journal: Carbohydrate polymers

Article Title: Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

doi: 10.1016/j.carbpol.2022.119547

Figure Lengend Snippet: C. neoformans EPS undergoes biophysical changes over time in solution. Native EPS (A) and lyophilized and reconstituted EPS (B) samples were examined by transmission electron microscopy (TEM) with negative staining at two levels of magnification. Native EPS contains a few aggregates as well as extracellular vesicles O(EVs), indicated with red arrows, while lyophilized and resuspended EPS contains many dense rosette-like structures previously reported for C. neoformans polysaccharides and glycogen . Native EPS and lyophilized and reconstituted samples kept in solution for 7, 14, or 28 days were examined by DLS (C). Lyophilized and resuspended samples have a larger particle size than native samples, judged by autocorrelation intensity of the scattered light as a function of particle diameter.

Article Snippet: C. neoformans serotype A strain H99 (ATCC 208821) cells were inoculated in Sabouraud rich medium from a frozen stock and grown for two days at 30 °C with agitation (150 rpm).

Techniques: Transmission Assay, Electron Microscopy, Negative Staining

Journal: Carbohydrate polymers

Article Title: Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

doi: 10.1016/j.carbpol.2022.119547

Figure Lengend Snippet: Lyophilization of C. neoformans EPS alters biological functions. Native EPS and lyophilized and resuspended samples were assayed for anti-GXM mAb binding by capture ELISA. Binding curves (A) of serially diluted mAb 18B7 as a function of EPS concentration in capture ELISA. Native EPS generally binds more strongly to the 2D10/18B7 capture/detection mAb pair than lyophilized EPS at a given mAb/antigen concentration. Statistical analysis (B) of binding in a capture ELISA double-array assay varying both EPS and mAb concentration shows that native EPS is statistically significantly better bound by anti-GXM mAbs than lyophilized EPS. **** p -value < 0.0001.

Article Snippet: C. neoformans serotype A strain H99 (ATCC 208821) cells were inoculated in Sabouraud rich medium from a frozen stock and grown for two days at 30 °C with agitation (150 rpm).

Techniques: Lyophilization, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Carbohydrate polymers

Article Title: Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

doi: 10.1016/j.carbpol.2022.119547

Figure Lengend Snippet: Structural model for the effects of lyophilization on C. neoformans EPS structure. EPS harvested from encapsulated C. neoformans is small, hydrated, rigid, and ordered when purified in its native form. Lyophilization causes alterations to the native form, resulting in disordered, condensed conformations that exclude water. Solvation of the lyophilized sample proceeds through gelation, wherein particles aquire a hydration shell but not structural waters. Over time in solution (28+ days) the condensed conformation will be lost and a polymer-like structure more similar to, but not the same as, the native structure is adopted.

Article Snippet: C. neoformans serotype A strain H99 (ATCC 208821) cells were inoculated in Sabouraud rich medium from a frozen stock and grown for two days at 30 °C with agitation (150 rpm).

Techniques: Lyophilization, Purification, Polymer

Journal: bioRxiv

Article Title: Bionized nanoferrite particles alter the course of experimental Cryptococcus neoformans pneumonia

doi: 10.1101/2021.12.21.473767

Figure Lengend Snippet: Higher recovery of lungs CFU 60 days after challenge with C. neoformans strain 24067 from the BNF-treated groups compared with the PBS control group ( A ). Intense immune reaction with numerous yeast organisms was seen following treatment with higher concentrations of nanoparticles in FVB/NJ mice. Representative histology images after 60 days of infection in PBS treated group ( B ), nanoparticle injection (5mgFe/mouse) before 24hrs ( C ) or after 24hrs ( D ) by H&E. It is possible to observe an increased number of yeast in both nanoparticle treated groups ( F, G ) compared to control ( E ) by PAS. Black arrows in E-G indicate yeast, red arrows indicates foamy macrophages and yellow indicates multinucleated giant cells. Blue arrow in F indicates organism inside a macrophage and green indicates neutrophils.

Article Snippet: C. neoformans var grubii serotype A strain H99 (ATCC 208821) was used in all A/J mice.

Techniques: Control, Infection, Injection

Journal: bioRxiv

Article Title: Bionized nanoferrite particles alter the course of experimental Cryptococcus neoformans pneumonia

doi: 10.1101/2021.12.21.473767

Figure Lengend Snippet: BNF 0.005 mg doses showed a significant decrease (p<0.05) of fungal CFU from recovered lungs ( A ) indicating a protective profile in lungs 14 days after challenge with C. neoformans strain 24067. H&E stained lung in FVB/NJ mice treated with PBS showing numerous macrophages and multinucleated giant cells (blue arrow) and neutrophils (green arrow) ( B, C ). BNF (0.005mg) treated lungs ( D, E ). PAS staining in PBS treated mouse ( F ) and in nanoparticle treated lungs ( G ). Tissues were stained with monoclonal antibody 18B7 conjugated with Oregon green with DAPI. Control PBS ( H ), BNF 0.5 mg ( I ), BNF 0.05 mg ( J ), BNF 0.005 mg ( K ). 10X magnification. Representative graph showing the IF-score generated by the IF slides ( L ).

Article Snippet: C. neoformans var grubii serotype A strain H99 (ATCC 208821) was used in all A/J mice.

Techniques: Staining, Control, Generated

Journal: bioRxiv

Article Title: Bionized nanoferrite particles alter the course of experimental Cryptococcus neoformans pneumonia

doi: 10.1101/2021.12.21.473767

Figure Lengend Snippet: A/J mice injected with lower doses of BNF showed a protective profile in lungs 14 days after challenge with C. neoformans strain H99. Mice injected with the lower doses of BNF nanoparticles (0.0005 mg) showed a survival of 100% in comparison to the control group (no BNF nanoparticle injection) with a survival of only 20% (p<0.05) ( A ). Lung fungal burden showed a significant decrease (p<0.05) in the number of recovered CFU in the mice that received the BNF 0.0005 mg ( B ). H&E stained lung sections showed numerous inflammatory cells surrounding Cryptococcus yeast (blue arrow) ( C, D ). BNF nanoparticle (0.0005mgFe/mouse) treated mouse ( E, F ). PAS staining showed presence of numerous Cryptococcus in PBS treated control mouse lung ( G , black arrow). BNF nanoparticle (0.0005mgFe) treated ( H ). Tissues were stained with monoclonal antibody 18B7 conjugated with Oregon green with DAPI (10x magnification). Control PBS ( I ), BNF 0.005 mg ( J ), BNF 0.0005 mg ( K ). Representative graph showing the IF-score generated by the IF slides ( L ). Lung and spleen cytokine levels during the course of infection ( M ). *p<0.05.

Article Snippet: C. neoformans var grubii serotype A strain H99 (ATCC 208821) was used in all A/J mice.

Techniques: Injection, Comparison, Control, Staining, Generated, Infection

Journal: bioRxiv

Article Title: Bionized nanoferrite particles alter the course of experimental Cryptococcus neoformans pneumonia

doi: 10.1101/2021.12.21.473767

Figure Lengend Snippet: Fungal burden from lungs and spleens of A/J mice 14 days after challenge with recently thawed C. neoformans strain H99 showed a slightly decrease from spleens of BNF 0.0005 mg group compared with PBS control groups ( A ). Lung and spleen cytokines levels were also analysed ( B ).

Article Snippet: C. neoformans var grubii serotype A strain H99 (ATCC 208821) was used in all A/J mice.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Hydroxylamine-armed fluorescent probe to study the distribution of reducing sugars in C. neoformans. A, synthesis of the R.E probe. B, proposed mechanism of action of the R.E armed fluorescent probe. The R.E probe reacts preferentially with aldehydes to form a stable oxime adduct that can be used to image the localization of reducing ends in the capsule and inside the cell. The hydroxylamine functionality (ONH2) is converted into an oxime (ON) after reacting with the reducing end glycan.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Incubation of hydroxylamine-armed fluorescent probe with C. neoformans. A, fluorescence spectrum of C. neoformans cells incubated with and without hydroxylamine-armed probe (R.E probe). Those that have been incubated with the R.E probe have increased emission maxima at ∼453 nm (excitation 360 nm) compared with encapsulated H99, CAP59Δ, or CAP67Δ with the exception of C. neoformans with encapsulated H99 with a melanized cell wall. Melanization of the cell wall is likely quenching the florescence of the probe. B, RFU of C. neoformans cells incubated with the R.E probe are significantly higher at emission maxima (440 nm) of the probe (excitation 360 nm). Error bars represent 95% confidence interval. Statistical significance was determined using unpaired t test (***, p ≤ 0.001; ****, p ≤ 0.0001). Experiments were performed in triplicate. C, encapsulated H99: the capsule shown by anti-GXM Mab 18B7-AF594 (red) displays no reactivity to the R.E probe 6 (green) and is localized at the cell wall–membrane interface. D, melanized C. neoformans cells display the same localization of the R.E probe. The R.E probe appears intracellularly possible in vesicular bodies and displays no reactivity toward the capsule, despite the melanization of the cell wall. E, acapsular mutant C. neoformans cap67Δ incubated with the R.E probe shows bright fluorescence intensity coming from the cytoplasm and the cell wall–membrane interface. Labeling of cytoplasm occurs in spherical vesicle-like structures in acapsular cap67Δ and melanized H99 cells. Scale, 5 μm.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Incubation, Fluorescence, Membrane, Mutagenesis, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Localization of reducing end polysaccharides is maintained across the Cryptococcus genus as labeled by the R.E probe. Incubations with the R.E probe (green), followed by India ink staining allows visualization of the capsule perimeter and where the reducing-ends reside. Any of the Cryptococcal sp. tested resulted in a similar staining pattern, suggesting that capsule architecture and biosynthesis are maintained across species. A, C. neoformans (ATCC 24067). B, C. albidus. C, C. gattii (ATCC 32608). D, C. gattii (ATCC 24065). E, incubation with the R.E probe increases RFU at emission of probe (excitation 360 nm). Error bars represent 95% confidence interval, Statistical significance was determined using unpaired t test (***, p ≤ 0.001; ****, p ≤ 0.0001). Experiments were performed in triplicate. Scale, 5 μm.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Labeling, Staining, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Capsule perturbation alters probe reactivity. H99 C. neoformans cells were grown in capsule-inducing minimal media for 3 days. Subsequently, the cells were independently processed with three different methods: DMSO, French press, and sonication. Thereafter, the cells were incubated with the fluorescent R.E probe (labels reducing carbohydrates, green) overnight, washed three times, stained for 30 min with Uvitex-2b (stains cell wall chitin, blue), and an 18B7-Alexa Fluor 594 conjugate (stains capsule, red) for 1 h. Scale, 10 μm.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Sonication, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Dynamic reorganization of C. neoformans polysaccharide capsule and cell–cell wall to facilitate budding. India ink staining perpendicular to emerging bud correlates with reorganization of chitin cell wall and capsule. 18B7 and R.E probe both have been polarized along the budding axis (line merge). This causes a change in capsule density perpendicular to the budding axis allowing India ink staining to penetrate deeper when gentle pressure is applied to the coverslip. Scale, 10 μm.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Staining, Gentle

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Exopolysaccharides of C. neoformans contain reducing ends. A, R.E probe was incubated with CPS, EPS (>10 kDa and >100 kDa), chitin (positive control), or alone for 24 h. The incubation was repeated in three independent experiments. Following dialysis, fluorescence emission spectra were recorded (excitation 360 nm) and compared with their nonincubated controls, showing an increase in fluorescence at the probe's emission maxima, indicating the R.E probe is forming a conjugate with the reducing end of various C. neoformans EPS and CPS isolates. Polysaccharides that were incubated with the R.E probe show >2-fold increase in RFU compared with controls, revealing that the shed exopolysaccharide contains reducing ends. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t test (****, p ≤ 0.0001). B, hypothesis: if reducing ends are present in the exopolysaccharide of C. neoformans, they would form stable conjugates with the R.E probe, which could be determined using a fluorescence spectrometer. Glycan notification followed the Symbol Nomenclature for Glycans (SNFG). C, 1H NMR analysis of >10- and >100-kDa exopolysaccharide isolates revealed the presence of aromatic peaks in the region of δ 7.7–8.6 ppm, which are not present in the original EPS fraction. Exploring C. neoformans with hydroxylamine-armed probe 28 aromatic signals are characteristic of the R.E probe. Characteristic structural reporter groups of anomeric mannose protons can be visualized from δ 5.5 to 4.9 ppm.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: Incubation, Positive Control, Fluorescence, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

doi: 10.1074/jbc.RA119.012251

Figure Lengend Snippet: Diagram illustrating three hypotheses for capsule assembly in C. neoformans. Polysaccharide molecules are shown in a linear manner for simplicity, with the localization of the reducing ends a key point for this depiction. The most parsimonious interpretation of the R.E. probe data supports hypothesis 1.

Article Snippet: Growth conditions C. neoformans serotype A strain H99 (American Type Culture Collection (ATCC) 208821), C. neoformans serotype D (ATCC 24067), C. gattii strains serotype B (ATCC 24065) and C (ATCC 32608), CAP59 Δ, CAP67 Δ, and C. albidus were grown for 48 h at 30 °C in capsule-inducing media composed of 10 m m MgSO 4 , 29.3 m m KH 2 PO 4 , 13 m m glycine, 3 μ m thiamine-HCl, adjusted to pH 5.5, and supplemented with 15 m m (regular minimal media) dextrose, with or without 1 m m l -DOPA (melanized cells).

Techniques: