scholarly journals Extracellular RNA as a potential activator of Neutrophil Extracellular Traps by Candida albicans biofilm

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Magdalena Smolarz ◽  
Marcin Zawrotniak ◽  
Dorota Satała ◽  
Maria Rapala-Kozik
Keyword(s):  
Candida Albicans ◽  
Human Neutrophils ◽  
Chromosomal Dna ◽  
Extracellular Rna ◽  
Rna Molecules ◽  
Whole Cells ◽  
Protective Environment ◽  
Migration Analysis ◽  
Biofilm Structure

Neutrophils represent the first line of innate host defense. The ability to inhibit the development of infections is associated with the involvement of several fighting strategies. The still poorly understood mechanism is netosis, involving the release of Extracellular Neutrophil Traps (NETs). NETs are complexes of chromosomal DNA and granule content. Such a web-like structure inhibits the spread of invaders. Netosis plays a significant role in combating Candida albicans infections. It has been shown that several factors, composing C. albicans cell surface mediate NETs production. However, the development of difficult to eradicate fungal infection is associated with the formation of the biofilm structure, which partially protects the pathogen cells from contact with the host’s immune system. One of the reasons for the creation of a such protective environment is the production of the extracellular matrix (ECM). The major components of the C. albicans ECM layer are lipids, proteins, carbohydrates but also extracellular nucleic acids, among which we observed a significant RNA content. Considering that the ECM consisting of RNA molecules is one of the first lines of contact between biofilms and neutrophils, our current studies aimed to assess the potential role of extracellular RNA in the triggering of the netosis process by human neutrophils in vitro. We showed that RNA purified from C. albicans biofilm structure and the whole cells have the capability to induction of ROS-dependent netosis pathway. Additionally, cell migration analysis indicate that RNA molecules may also be an effective chemotactic agent. This work was supported by NCN (2019/33/B/NZ6/02284).

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

2019 ◽  
Vol 44 (6) ◽  
pp. 810-821
Author(s):  
Edibe Avci ◽  
Yeliz Z. Akkaya-Ulum ◽  
Digdem Yoyen-Ermis ◽  
Gunes Esendagli ◽  
Banu Balci-Peynircioglu
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Neutrophil Migration ◽  
Cost Effective ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

scholarly journals Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps

2021 ◽  
Vol 11 ◽  
Author(s):  
Magdalena Smolarz ◽  
Marcin Zawrotniak ◽  
Dorota Satala ◽  
Maria Rapala-Kozik
Keyword(s):  
Candida Albicans ◽  
Nucleic Acids ◽  
Fungal Infection ◽  
Fungal Infections ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
High Concentration ◽  
First Line ◽  
Extracellular Traps ◽  
Biofilm Structure

Neutrophils, the first line of the host’s defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

scholarly journals Release of Candida albicans yeast antigens upon interaction with human neutrophils in vitro

1997 ◽  
Vol 46 (9) ◽  
pp. 747-755 ◽  
Author(s):  
C. ASHLEY ◽  
M. MORHART ◽  
R. RENNIE ◽  
B. ZIOLA
Keyword(s):  
Candida Albicans ◽  
Human Neutrophils

IMMUNOMODULATORY ACTIVITY OF METHANOLIC EXTRACTS OF SEEDS AND BARK OF PONGAMIA GLABRA VENT. ON HUMAN NEUTROPHILS

INDIAN DRUGS ◽  
2013 ◽  
Vol 50 (03) ◽  
pp. 46-52
Author(s):  
S.S. Heroor ◽  
◽  
A.V. Beknal ◽  
N.K. Mahurkar
Keyword(s):  
Candida Albicans ◽  
In Vitro Models ◽  
Nitroblue Tetrazolium ◽  
Human Neutrophils ◽  
Immunomodulatory Activity ◽  
Chemotaxis Assay ◽  
Methanolic Extracts ◽  
Pongamia Glabra

Methanolic extracts of seeds and bark of Pongamia glabra Vent. (250 mg/kg and 500 mg/kg p.o.) in the concentration range 100, 50, 25, 12 and 6.25 µg were subjected to evaluate the phagocytic effect on human neutrophils using the in-vitro models – nitroblue tetrazolium (NBT) dye test, phagocyotosis of Candida albicans and chemotaxis assay. The extracts of the plant in the concentration range 100,50,25,12 and 6.25 µg showed significant (P <0.01) phagocytic effect on human neutrophils in the parameters studied. Methanolic extracts of seeds and barks of Pongamia glabra Vent. exhibited immunostimulant property in in-vitro models.

scholarly journals Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene

1998 ◽  
Vol 66 (5) ◽  
pp. 1953-1961 ◽  
Author(s):  
Deborah R. Wysong ◽  
Laurent Christin ◽  
Alan M. Sugar ◽  
Phillips W. Robbins ◽  
Richard D. Diamond
Keyword(s):  
Candida Albicans ◽  
Parental Strain ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Nucleotide Sequence Analysis ◽  
Infection Model ◽  
Null Mutant ◽  
Multiple Alleles ◽  
Catalase Gene

ABSTRACT Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a singleC. albicans-derived catalase gene, designatedCAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicansATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation usingURA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

scholarly journals Deletion of the Two-Component Histidine Kinase Gene (CHK1) of Candida albicans Contributes to Enhanced Growth Inhibition and Killing by Human Neutrophils In Vitro

2002 ◽  
Vol 70 (2) ◽  
pp. 985-987 ◽  
Author(s):  
Antonella Torosantucci ◽  
Paola Chiani ◽  
Flavia De Bernardis ◽  
Antonio Cassone ◽  
Jose Antonio Calera ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Histidine Kinase ◽  
Polymorphonuclear Leukocytes ◽  
Human Neutrophils ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Killing Effect ◽  
Growth Inhibitory ◽  
Two Component

ABSTRACT We have observed that human neutrophils (polymorphonuclear leukocytes [PMNs]) have an increased growth-inhibitory and killing effect on a strain of Candida albicans with a deletion of CHK1, a gene encoding a putative histidine kinase. The PMN effect was not due to increased phagocytosis of the null strain. This observation may partially explain the reduced virulence in a hematogenously disseminated murine model of candidiasis.

Effect of cerulenin and sodium butyrate on chitin synthesis in Candida albicans

1987 ◽  
Vol 33 (6) ◽  
pp. 546-550 ◽  
Author(s):  
Phyllis C. Braun ◽  
Richard F. Hector ◽  
Michael E. Kamark ◽  
John T. Hart ◽  
Ronald L. Cihlar
Keyword(s):  
Candida Albicans ◽  
Sodium Butyrate ◽  
Marked Decrease ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Time Intervals ◽  
Whole Cells ◽  
Insoluble Material ◽  
Membrane Bound

Experiments were conducted to gain insight concerning the mechanism(s) whereby cerulenin and sodium butyrate affect chitin synthesis in Candida albicans. In vitro studies with isolated membrane-bound chitin synthase from C. albicans, strain 4918, showed that neither agent affected the level of either unactivated or trypsin-activated enzyme activity. Subsequent studies utilizing protoplasts revealed that early in the cell wall regeneration process, cells treated with cerulenin or butyrate synthesized chitin at a rate equal to untreated controls, as measured by the incorporation of [3H]-N-acetylglucosamine (GlcNAc) into acid–alkali insoluble material. However, after 40 min of incubation, the incorporation of [3H]GlcNAc into chitin is reduced in cells treated with either agent. On the other hand, samples taken during the same time intervals and analyzed by flow cytometry suggested that the amount of chitin synthesis in treated and untreated cells was identical. A marked decrease in fluorescence was observed in similar experiments using polyoxin D, a direct inhibitor of chitin synthase activity. Experiments that measured uptake of [3H]GlcNAc into both whole cells and protoplasts demonstrated that cerulenin and butyrate had no effect on the transport of the chitin precursor.

Aktivitas Antifungi Air Perasan Bawang Merah (Allium ascalonicum L.) Terhadap Candida albicans Secara In Vitro

2017 ◽  
Vol 9 (2) ◽  
pp. 71
Author(s):  
Nurhasanah Nurhasanah ◽  
Fauzia Andrini ◽  
Yulis Hamidy
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Allium Sativum ◽  
Fungicidal Activity ◽  
Positive Control ◽  
Negative Control ◽  
Randomized Design ◽  
Significant Difference ◽  
Completely Randomized Design

Shallot (Allium ascalonicum L.) has been known as traditional medicine. Shallot which has same genus with garlic(Allium sativum L.) contains allicin that is also found in garlic and has been suspected has fungicidal activity toCandida albicans. It is supported by several researches. Therefore, shallot is suspected has antifungal activity too.The aim of this research was to know antifungal activity of shallot’s water extortion againsts Candida albicans invitro. This was a laboratory experimental research which used completely randomized design, with diffusion method.Shallot’s water extortion was devided into three concentrations, there were 50%, 100% and 200%. Ketoconazole 2%was positive control and aquadest was negative control. The result of this research based on analysis of varians(Anova), there was significant difference between several treatments and was confirmed with Duncan New MultipleRange Test (DNMRT) p<0,05, there was significant difference between 100% shallot’s water extortion with othertreatments, but there was no significant difference between 50% shallot’s water extortion with 200% shallot’s. Theconclusion was shallot’s water extortion had antifungal activity againsts Candida albicans with the best concentration100%, but it was lower than ketoconazole 2%.

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