scholarly journals Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape

2021 ◽  
Vol 10 ◽  
Author(s):  
Yohan Ricci Zonta ◽  
Ana Laura Ortega Dezen ◽  
Amanda Manoel Della Coletta ◽  
Kaio Shu Tsyr Yu ◽  
Larissa Carvalho ◽  
...  
Keyword(s):  
Paracoccidioides Brasiliensis ◽  
Polymorphonuclear Neutrophils ◽  
Fungal Genome ◽  
Dna Structures ◽  
Escape Mechanism ◽  
Virulent Isolate ◽  
Human Pmns ◽  
Etiological Agents ◽  
Relative Gene

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

scholarly journals Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan

2018 ◽  
Vol 11 (4) ◽  
pp. 330-346 ◽  
Author(s):  
João Alfredo Moraes ◽  
Ana Clara Frony ◽  
Pedro Barcellos-de-Souza ◽  
Marcel Menezes da Cunha ◽  
Thayanne Brasil Barbosa Calcia ◽  
...  
Keyword(s):  
Polymorphonuclear Neutrophils ◽  
In Vivo Experiments ◽  
Formyl Peptide ◽  
Human Pmns ◽  
Oxidative Effects ◽  
Species Production ◽  
Bacterial Products

Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells.

scholarly journals 43 kDa Glycoprotein (gp43) from Paracoccidioides brasiliensis Induced IL-17A and PGE2 Production by Human Polymorphonuclear Neutrophils: Involvement of TLR2 and TLR4

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Taiane Priscila Gardizani ◽  
Amanda Manoel Della Coletta ◽  
Graziela Gorete Romagnoli ◽  
Rosana Puccia ◽  
Ana Paula Moreira Serezani ◽  
...  
Keyword(s):  
Paracoccidioides Brasiliensis ◽  
Polymorphonuclear Neutrophils ◽  
Host Susceptibility ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tlr4 Expression ◽  
Adhesive Properties ◽  
Healthy Human ◽  
Surface Receptors ◽  
Etiologic Agents

The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.

scholarly journals Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Mariane C. Bagatin ◽  
Arethusa L. Pimentel ◽  
Débora C. Biavatti ◽  
Ernani A. Basso ◽  
Erika S. Kioshima ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Fungal Infections ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungi ◽  
Dynamics Simulation ◽  
Homoserine Dehydrogenase ◽  
Content Type ◽  
Zinc Database ◽  
Etiological Agents

ABSTRACT This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 μg · ml−1, respectively, for the Pb18 isolate of P. brasilensis, 64 μg · ml−1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

In vitro effects of non-steroidal anti-inflammatory drugs (Meloxicam and Flunixin Meglumine) and phytochemical (Harpagoside) on the respiratory burst of porcine polymorphonuclear neutrophils (PMNs)

2021 ◽  
Vol 1 (2) ◽  
pp. 8-12
Author(s):  
Giuseppe Marruchella ◽  
Francesco Mosca ◽  
Jasmine Hattab ◽  
Abigail R. Trachtman ◽  
Pietro G. Tiscar
Keyword(s):  
Veterinary Medicine ◽  
Respiratory Burst ◽  
Polymorphonuclear Neutrophils ◽  
Flunixin Meglumine ◽  
Human Pmns ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
In Vitro Effects

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in veterinary medicine. Additionally, interest regarding the anti-infammatory properties of phytochemicals has emerged in recent years. In the present study, we aim to investigate the in vitro effects of meloxicam, flunixin meglumine, and harpagoside on the respiratory burst of porcine polymorphonuclear neutrophils (PMNs). We observed that harpagoside was able to suppress the respiratory burst, similarly to flunixin meglumine. Conversely, meloxicam enhanced the PMNs response. However, these effects were only detected at concentrations higher than those achievable in plasma and tissues. The present study intends to offer insights into the role of these molecules on phagocytosis mechanisms in animals to complement what is already known regarding human PMNs.

Superoxide released from neutrophils causes a reduction in nitric oxide gas

1998 ◽  
Vol 275 (6) ◽  
pp. L1120-L1126 ◽  
Author(s):  
Kimberly L. Jones ◽  
Ty W. Bryan ◽  
Patricia A. Jinkins ◽  
Keith L. Simpson ◽  
Matthew B. Grisham ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Distress Syndrome ◽  
Polymorphonuclear Neutrophils ◽  
Epithelial Cell Line ◽  
Lung Epithelial ◽  
Human Pmns

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Discrimination of monomer from multimer DNA disk-shaped toruses by freezeetch TEM

1984 ◽  
Vol 42 ◽  
pp. 210-211
Author(s):  
George C. Ruben ◽  
Kenneth A. Marx
Keyword(s):  
Biochemical Data ◽  
Dna Structures ◽  
Ice Surface ◽  
Double Stranded Dna ◽  
Data Support ◽  
Dna Organization ◽  
Trivalent Cations

In vitro collapse of DNA by trivalent cations like spermidine produces torus (donut) shaped DNA structures thought to have a DNA organization similar to certain double stranded DNA bacteriophage and viruses. This has prompted our studies of these structures using freeze-etch low Pt-C metal (9Å) replica TEM. With a variety of DNAs the TEM and biochemical data support a circumferential DNA winding model for hydrated DNA torus organization. Since toruses are almost invariably oriented nearly horizontal to the ice surface one of the most accessible parameters of a torus population is annulus (ring) thickness. We have tabulated this parameter for populations of both nicked, circular (Fig. 1: n=63) and linear (n=40: data not shown) ϕX-174 DNA toruses. In both cases, as can be noted in Fig. 1, there appears to be a compact grouping of toruses possessing smaller dimensions separated from a dispersed population possessing considerably larger dimensions.

scholarly journals Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment

2021 ◽  
Vol 14 (3) ◽  
pp. 220
Author(s):  
Claudia Taborda Gómez ◽  
Fabiana Lairion ◽  
Marisa Repetto ◽  
Miren Ettcheto ◽  
Amalia Merelli ◽  
...  
Keyword(s):  
Refractory Epilepsy ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Nuclear Condensation ◽  
Concentration Dependent Manner ◽  
Superoxide Anion Production ◽  
Excretory Function ◽  
Psychoactive Effects ◽  
Stress Damage

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti-epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1-CB2) or multiple non-canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P-glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration-dependent manner, while low CBD concentration decreased PMN viability in a time-dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti-inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.

scholarly journals DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1357
Author(s):  
Rubén Torres ◽  
Carolina Gándara ◽  
Begoña Carrasco ◽  
Ignacio Baquedano ◽  
Silvia Ayora ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Stable Complex ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Structures ◽  
Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

scholarly journals In Vitro Anti-Inflammatory and Immunomodulatory Activities of an Extract from the Roots of Bupleurum rotundifolium

Medicines ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 101 ◽  
Author(s):  
Cholet ◽  
Decombat ◽  
Vareille-Delarbre ◽  
Gainche ◽  
Berry ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Polymorphonuclear Neutrophils ◽  
Ros Production ◽  
Peripheral Blood Mononuclear ◽  
Aerial Parts ◽  
Bupleurum Scorzonerifolium Willd ◽  
Anti Inflammatory ◽  
Inflammatory Profile ◽  
Bupleurum Scorzonerifolium

Background: Some Bupleurum species, such as the Bupleurum chinense DC. or the Bupleurum scorzonerifolium Willd have been extensively studied (especially their roots) for the treatment of inflammation. In contrast, only compounds extracted from the aerial parts of Bupleurum rotundifolium have been studied and showed anti-inflammatory or antiproliferative activities. This study was conducted to investigate the antioxidant, anti-inflammatory, and immunomodulatory effects of Bupleurum rotundifolium roots. Methods: To tackle the various aspects of inflammation, we studied in vitro a methanolic extract from the roots of Bupleurum rotundifolium on peripheral blood mononuclear cells (PBMCs), polymorphonuclear neutrophils (PMNs), and the monocytic cells THP-1. Its antioxidant capacities and iron-chelating activity were assessed. The extract was tested on THP-1 differentiation, reactive oxygen species (ROS) production by leukocytes, neutrophils chemotaxis, cytokines, PGE2 production, and NF-κB activation in PBMCs. Results: The extract showed a decreased ROS production in stimulated cells. It increased PBMC chemokine secretion and up-regulated the differentiation of THP-1 monocytes into macrophage-like cells, indicating a potential interest of the extract in the resolution of acute inflammation. In addition, the analysis of cytokine production suggests that Bupleurum rotundifolium has immunomodulatory properties. Conclusions: Cytokines secretion, especially IL-1β and IL-12p70, provided us with a set of indicators suggesting that the extract might be able to drive the polarization of macrophages and lymphocytes toward a Th2 anti-inflammatory profile in excessive inflammation.

scholarly journals The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

2012 ◽  
Vol 45 (6) ◽  
pp. 739-744 ◽  
Author(s):  
Francisco Laurindo da Silva ◽  
Raphael Sanzio Pimenta ◽  
Juliana Fonseca Moreira da Silva ◽  
Déborah Aparecida Negrão Corrêa ◽  
Ary Corrêa Junior
Keyword(s):  
Epithelial Cell ◽  
Paracoccidioides Brasiliensis ◽  
Yeast Cells ◽  
Epithelial Cell Line ◽  
Con A ◽  
Culture Methods ◽  
Disease Treatment ◽  
Adhesion Behavior ◽  
And Control

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

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