scholarly journals Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

2019 ◽  
Author(s):  
Conor J. Crawford ◽  
Radamés J.B. Cordero ◽  
Lorenzo Guazzelli ◽  
Maggie P. Wear ◽  
Anthony Bowen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Probe ◽  
Chemical Biology ◽  
Membrane Integrity ◽  
The Body ◽  
Fluorescence Signal ◽  
Initiation Point ◽  
Cell Membrane Integrity ◽  
Capsule Assembly

ABSTRACTChemical biology is an emerging field that allows the study and manipulation of biological systems using probes that inform on structure based on their reactivity. We report the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the Cryptococcus neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing end glycans at this location where the capsule attachment to the cell body occurs. In contrast, there was no fluorescence signal in the body of the capsule. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear-stress resulted in capsule alterations that affected the binding of the probe as reducing ends were exposed, and cell membrane integrity was compromised. In contrast to the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly where reducing ends localize to the cell wall surface, supporting previous work suggesting that this is an initiation point for capsular assembly. Chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides.

scholarly journals Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

2020 ◽  
Vol 295 (13) ◽  
pp. 4327-4340 ◽  
Author(s):  
Conor J. Crawford ◽  
Radamés J. B. Cordero ◽  
Lorenzo Guazzelli ◽  
Maggie P. Wear ◽  
Anthony Bowen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Probe ◽  
Chemical Biology ◽  
Membrane Integrity ◽  
Fluorescence Signal ◽  
Fungal Virulence ◽  
Initiation Point ◽  
Cell Membrane Integrity ◽  
Capsule Assembly

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall–membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.

Electrical Impedance Tomography of Cell Viability in Tissue With Application to Cryosurgery

2004 ◽  
Vol 126 (2) ◽  
pp. 305-309 ◽  
Author(s):  
Rafael Davalos ◽  
Boris Rubinsky
Keyword(s):  
Electrical Properties ◽  
Electrical Impedance Tomography ◽  
Electrical Impedance ◽  
Cell Damage ◽  
Membrane Integrity ◽  
The Body ◽  
Impedance Tomography ◽  
Cell Membrane Integrity ◽  
Minimally Invasive Surgical Procedure ◽  
Minimally Invasive Surgical

Tissue damage that is associated with the loss of cell membrane integrity should alter the bulk electrical properties of the tissue. This study shows that electrical impedance tomography (EIT) should be able to detect and image necrotic tissue inside the body due to the permeabilization of the membrane to ions. Cryosurgery, a minimally invasive surgical procedure that uses freezing to destroy undesirable tissue, was used to investigate the hypothesis. Experimental results with liver tissue demonstrate that cell damage during freezing results in substantial changes in tissue electrical properties. Two-dimensional EIT simulations of liver cryosurgery, which employ the experimental data, demonstrate the feasibility of this application.

scholarly journals Antimicrobial Peptide AMP-17 Affects Candida albicans by Disrupting Its Cell Wall and Cell Membrane Integrity

2020 ◽  
Vol Volume 13 ◽  
pp. 2509-2520
Author(s):  
Huiling Ma ◽  
Xinyu Zhao ◽  
Longbing Yang ◽  
Peipei Su ◽  
Ping Fu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Membrane ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Cell Membrane Integrity

scholarly journals Altering the Proclivity towards Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Using Combinations with Other Antibiotics

2012 ◽  
Vol 56 (10) ◽  
pp. 5046-5053 ◽  
Author(s):  
Andrew D. Berti ◽  
Justine E. Wergin ◽  
Gary G. Girdaukas ◽  
Scott J. Hetzel ◽  
George Sakoulas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Membrane ◽  
Membrane Integrity ◽  
Wall Thickening ◽  
Methicillin Resistant ◽  
Content Type ◽  
Cell Membrane Integrity ◽  
Cell Membrane Fluidity

ABSTRACTDaptomycin (DAP) is increasingly used as a part of combination therapy, particularly in complex methicillin-resistantStaphylococcus aureus(MRSA) infections. While multiple studies have reported the potential for synergy between DAP and adjunctive anti-infectives, few have examined the influence of adjunctive therapy on the emergence of DAP resistance. This study examined eight adjunctive antimicrobial combinations with DAPin vitroand the emergence of DAP resistance over time (up to 4 weeks) using clinical isolates of DAP-susceptible MRSA (MIC, 0.5 μg/ml) in which DAP resistance subsequently developed during patient therapy (MIC, 3 μg/ml). In addition to DAP susceptibility testing, selected strains were examined for phenotypic changes associated with DAP resistance, including changes to cell wall thickness (CWT) and cell membrane alterations. The addition of either oxacillin or clarithromycin in medium containing DAP significantly inhibited the development of DAP resistance through the entirety of the 4-week exposure (10- to 32-fold MIC reduction from that of DAP alone). Combinations with rifampin or fosfomycin were effective in delaying the emergence of DAP resistance through the end of week one only (week one MIC, 0.5 μg/ml; week four MIC, 24 μg/ml). Cell wall thickening was observed for all antibiotic combinations regardless of their effect on the DAP MIC (14 to 70% increase in CWT), while changes in cell membrane fluidity were variable and treatment dependent. DAP showed reduced activity against strains with DAP MICs of 1 to 12 μg/ml, but cell membrane integrity was still disrupted at concentrations achieved with doses greater than 10 mg/kg of body weight. The emergence of DAP resistance in MRSA is strongly influenced by the presence of subinhibitory concentrations of adjunctive antimicrobials. These data suggest that combining DAP with oxacillin or clarithromycin may delay the development of DAP resistance in cases requiring prolonged antibiotic therapy.

scholarly journals Requirement of a Tsp2-Type Tetraspanin for Laccase Repression and Stress Resistance in the Basidiomycete Cryptococcus neoformans

2011 ◽  
Vol 78 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Zhongming Li ◽  
Jiannan Bi ◽  
Jiao Yang ◽  
Jiao Pan ◽  
Zhixiong Sun ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Stress Resistance ◽  
Glucose Repression ◽  
Membrane Integrity ◽  
Content Type ◽  
Cell Membrane Integrity ◽  
Underlying Basis ◽  
Dependent Protein ◽  
Pka Pathway ◽  
First Time

ABSTRACTFungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene,TSP2-1, was required for laccase repression by glucose in the basidiomyceteCryptococcus neoformans.TSP2-1encodes a Tsp2-type tetraspanin. The disruption ofTSP2-1resulted in constant melanin formation and the expression of the laccase geneLAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutanttsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles ofTSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression inC. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.

scholarly journals Octominin: A Novel Synthetic Anticandidal Peptide Derived from Defense Protein of Octopus minor

Marine Drugs ◽  
2020 ◽  
Vol 18 (1) ◽  
pp. 56 ◽  
Author(s):  
Chamilani Nikapitiya ◽  
S.H.S. Dananjaya ◽  
H.P.S.U. Chandrarathna ◽  
Mahanama De Zoysa ◽  
Ilson Whang
Keyword(s):  
Cell Wall ◽  
Cell Membrane ◽  
Antimicrobial Agents ◽  
Membrane Integrity ◽  
Alpha Helix ◽  
Infection Model ◽  
Dependent Manner ◽  
Cell Membrane Integrity ◽  
Defense Protein ◽  
Octopus Minor

The rapid emergence of multidrug-resistant pathogens makes an urgent need for discovering novel antimicrobial agents as alternatives to conventional antibiotics. Towards this end, we designed and synthesized a synthetic peptide of 23 amino acids (AAs) (1GWLIRGAIHAGKAIHGLIHRRRH23) from a defense protein 3 cDNA sequence of Octopus minor. The sequence of the peptide, which was named Octominin, had characteristic features of known antimicrobial peptides (AMPs) such as a positive charge (+5), high hydrophobic residue ratio (43%), and 1.86 kcal/mol of Boman index. Octominin was predicted to have an alpha-helix secondary structure. The synthesized Octominin was 2625.2 Da with 92.5% purity. The peptide showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 50 and 200 μg/mL, respectively, against Candida albicans. Field emission scanning electron microscopy observation confirmed that Octominin caused ultrastructural cell wall deformities in C. albicans. In addition, propidium iodide penetrated the Octominin-treated C. albicans cells, further demonstrating loss of cell membrane integrity that caused cell death at both MIC and MFC. Octominin treatment increased the production of intracellular reactive oxygen species and decreased cell viability in a concentration dependent manner. Cytotoxicity assays revealed no significant influence of Octominin on the viability of human embryonic kidney 293T cell line, with over 95% live cells in the Octominin-treated group observed up to 100 µg/mL. Moreover, we confirmed the antifungal action of Octominin in vivo using a zebrafish experimental infection model. Overall, our results demonstrate the Octominin is a lead compound for further studies, which exerts its effects by inducing cell wall damage, causing loss of cell membrane integrity, and elevating oxidative stress.

scholarly journals Vesicular Polysaccharide Export in Cryptococcus neoformans Is a Eukaryotic Solution to the Problem of Fungal Trans-Cell Wall Transport

2006 ◽  
Vol 6 (1) ◽  
pp. 48-59 ◽  
Author(s):  
Marcio L. Rodrigues ◽  
Leonardo Nimrichter ◽  
Débora L. Oliveira ◽  
Susana Frases ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Lipid Vesicles ◽  
Central Question ◽  
Major Virulence Factor ◽  
Animal Infection ◽  
Capsule Assembly ◽  
Extracellular Environment

ABSTRACT The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.

scholarly journals The Vacuolar Morphogenesis Protein Vam6-Like Protein Vlp1 Is Required for Pathogenicity of Cryptococcus neoformans

2021 ◽  
Vol 7 (6) ◽  
pp. 418
Author(s):  
Cheng-Li Fan ◽  
Tong-Bao Liu
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Critical Role ◽  
Membrane Integrity ◽  
Infection Model ◽  
Fungal Virulence ◽  
Cell Membrane Integrity ◽  
Fungal Meningitis ◽  
Virulence Attenuation ◽  
F Box Protein

Cryptococcus neoformans is an encapsulated yeast pathogen that infects immunocompromised patients to cause fungal meningitis, resulting in hundreds of thousands of deaths each year. F-box protein Fbp1, the key component of the E3 ubiquitin ligase, plays a critical role in fungal development and virulence in fungal pathogens. In this study, we identified a potential substrate of Fbp1, the vacuolar morphogenesis protein Vam6-like protein Vlp1, and evaluated its role in virulence in C. neoformans. Deletion or overexpression of the VLP1 gene results in abnormal capsule formation and melanin production of C. neoformans. Stress tolerance assay showed that the vlp1Δ mutant was sensitive to SDS and NaCl but not to CFW or Congo red, indicating that Vlp1 might regulate the cell membrane integrity in C. neoformans. Fungal virulence assay showed that Vlp1 was essential for the pathogenicity of C. neoformans, as vlp1Δ mutants are avirulent in the mouse systematic infection model of cryptococcosis. The progression of fungal infection revealed that the vlp1Δ mutants were gradually eliminated from the lungs of the mice after infection. Moreover, the vlp1Δ mutants showed a proliferation defect inside macrophages and a viability defect in the host complement system, which likely contributes to the virulence attenuation of the vlp1Δ mutants. In summary, our results revealed that the vacuolar morphogenesis protein Vam6-like protein Vlp1 is essential for the pathogenicity of C. neoformans.

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