scholarly journals Evidence for an Intramacrophage Growth Phase of Mycobacterium ulcerans

2006 ◽  
Vol 75 (2) ◽  
pp. 977-987 ◽  
Author(s):  
Egídio Torrado ◽  
Alexandra G. Fraga ◽  
António G. Castro ◽  
Pieter Stragier ◽  
Wayne M. Meyers ◽  
...  
Keyword(s):  
Buruli Ulcer ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Host Cells ◽  
Delayed Type Hypersensitivity ◽  
In Vitro Assays ◽  
Mouse Macrophages ◽  
Inflammatory Infiltrates ◽  
Extracellular Location

ABSTRACT Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.

scholarly journals Aberrant stromal tissue factor and mycolactone-driven vascular permeability, exacerbated by IL-1β, orchestrate pathogenic fibrin formation in Buruli ulcer lesions

2021 ◽  
Author(s):  
Louise Tzung-Harn Hsieh ◽  
Scott J Dos Santos ◽  
Joy Ogbechi ◽  
Aloysius D Loglo ◽  
Francisco J Salguero ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
In Vitro Assays ◽  
Platelet Glycoprotein ◽  
Fibrin Deposition ◽  
Clotting Factors ◽  
Extrinsic Pathway ◽  
Fibrin Formation

The neglected tropical disease Buruli ulcer, caused by Mycobacterium ulcerans infection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to the M. ulcerans exotoxin mycolactone depletes the expression of thrombomodulin and impacts anticoagulation at the endothelial cell surface. Moreover, while widespread fibrin deposition is a common feature of BU lesions, the cause of this phenotype is not clear. Here, we performed sequential staining of serial tissue sections of BU patient biopsies and unbiased analysis of up to 908 individual non-necrotic vessels of eight BU lesions to investigate its origins. Most vessels showed evidence of endothelial dysfunction being thrombomodulin-negative, von Willebrand factor-negative and/or had endothelium that stained positively for tissue factor (TF). Primary haemostasis was rarely evident by platelet glycoprotein CD61 staining. Localisation of TF in these lesions was complex and aberrant, including diffuse staining of the stroma some distance from the basement membrane and TF-positive infiltrating cells (likely eosinophils). This pattern of abnormal TF staining was the only phenotype that was significantly associated with fibrin deposition, and its extent correlated significantly with the distance that fibrin deposition extended into the tissue. Hence, fibrin deposition in Buruli ulcer lesions is likely driven by the extrinsic pathway of coagulation. To understand how this could occur, we investigated whether clotting factors necessary for fibrin formation might gain access to the extravascular compartment due to loss of the vascular barrier. In vitro assays using primary vascular and lymphatic endothelial cells showed that mycolactone increased the permeability of monolayers to dextran within 24 hours. Moreover, co-incubation of cells with interleukin-1β exacerbated mycolactones effects, nearly doubling the permeability of the monolayer compared to each challenge alone. We propose that leaky vascular and lymphatic systems are important drivers of extravascular fibrin deposition, necrosis and oedema frequently seen in Buruli ulcer patients.

scholarly journals 1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1053
Author(s):  
Lidia Węglińska ◽  
Adrian Bekier ◽  
Katarzyna Dzitko ◽  
Barbara Pacholczyk-Sienicka ◽  
Łukasz Albrecht ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Direct Action ◽  
Human Fibroblasts ◽  
Imidazole Ring ◽  
Host Cells ◽  
In Vitro Assays ◽  
Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

scholarly journals Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening

2018 ◽  
Author(s):  
María Pilar Arenaz Callao ◽  
Rubén González del Río ◽  
Ainhoa Lucía Quintana ◽  
Charles J. Thompson ◽  
Alfonso Mendoza-Losana ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Amoxicillin Clavulanate ◽  
Low Toxicity ◽  
Potential Use ◽  
Ulcer Treatment

ABSTRACTThe potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

scholarly journals Molecular Mechanisms Underpinning the Circulation and Cellular Uptake of Mycobacterium ulcerans Toxin Mycolactone

2021 ◽  
Vol 12 ◽  
Author(s):  
Bruno Tello Rubio ◽  
Florence Bugault ◽  
Blandine Baudon ◽  
Bertrand Raynal ◽  
Sébastien Brûlé ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Molecular Mechanisms ◽  
Scavenger Receptor ◽  
Buruli Ulcer ◽  
Systemic Circulation ◽  
Mycobacterium Ulcerans ◽  
Host Cells ◽  
Ulcer Disease ◽  
Major Mechanism ◽  
Specific Association

Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.

scholarly journals First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

2019 ◽  
Author(s):  
Tchalare Kondi Makagni ◽  
Maman Issaka ◽  
Piten Ebekalisai ◽  
Disse Kodjo ◽  
Essossimna A. Kadanga ◽  
...  
Keyword(s):  
Fine Needle Aspiration ◽  
Slow Growth ◽  
Buruli Ulcer ◽  
Needle Aspiration ◽  
Mycobacterium Ulcerans ◽  
Clinical Samples ◽  
Direct Examination ◽  
Fine Needle ◽  
Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

scholarly journals Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

2021 ◽  
Author(s):  
Agustina P. Bertolin ◽  
Florian Weissmann ◽  
Jingkun Zeng ◽  
Viktor Posse ◽  
Jennifer C. Milligan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Etiologic Agent ◽  
Host Cells ◽  
Rdrp Activity ◽  
Chemical Library ◽  
Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

scholarly journals Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

2009 ◽  
Vol 75 (17) ◽  
pp. 5667-5675 ◽  
Author(s):  
Michael K�ser ◽  
Julia Hauser ◽  
Pamela Small ◽  
Gerd Pluschke
Keyword(s):  
Buruli Ulcer ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Nucleotide Polymorphisms ◽  
Differentiation Markers ◽  
Single Nucleotide ◽  
Virulence Plasmids ◽  
Sequence Polymorphisms ◽  
Genetic Features ◽  
Relationship Of

ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

scholarly journals In Vitro Killing of Mycobacterium ulcerans by Acidified Nitrite

2004 ◽  
Vol 48 (8) ◽  
pp. 3130-3132 ◽  
Author(s):  
R. Phillips ◽  
S. Kuijper ◽  
N. Benjamin ◽  
M. Wansbrough-Jones ◽  
M. Wilks ◽  
...  
Keyword(s):  
Buruli Ulcer ◽  
Mycobacterium Ulcerans

ABSTRACT Mycobacterium ulcerans, which causes Buruli ulcer, was exposed to acidified nitrite or to acid alone for 10 or 20 min. Killing was rapid, and viable counts were reduced below detectable limits within 10 min of exposure to 40 mM acidified nitrite. M. ulcerans is highly susceptible to acidified nitrite in vitro.

scholarly journals Molecular Docking Studies of Curcumin Analogues against SARS-CoV-2 Spike Protein

2021 ◽  
Author(s):  
Jéssica Nogueira ◽  
Flávia Verza ◽  
Felipe Nishimura ◽  
Umashankar Das ◽  
Ícaro Caruso ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Virus Disease ◽  
Etiologic Agent ◽  
Host Cells ◽  
Spike Protein ◽  
Therapeutic Effects ◽  
Host Cell Membrane ◽  
Curcumin Analogues

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the etiologic agent of the current pandemic of corona virus disease 2019 (COVID-19) that has inflicted the loss of thousands of lives worldwide. The coronavirus surface spike (S) glycoprotein is a class I fusion with a S1 domain which is attached to the human angiotensin converting enzyme 2 (ACE2) receptor, and a S2 domain which enables fusion with the host cell membrane and internalization of the virus. Curcumin has been suggested as a potential drug to control inflammation and as a potential inhibitor of S protein, but its therapeutic effects are hampered by poor bioavailability. We performed a molecular docking and dynamic study using 94 curcumin analogues designed to have improved metabolic stability against the SARS-CoV-2 spike protein and compared their affinity with curcumin and other potential inhibitors. The docking analysis suggested that the S2 domain is the main target of these compounds and compound 2606 displayed a higher binding affinity (-9.6 kcal mol-1) than curcumin (-6.8 kcal mol-1) and the Food and Drug Administration (FDA) approved drug hydroxychloroquine (-6.3 kcal mol-1). Further additional validation in vitro and in vivo of these compounds against SARS-CoV-2 may provide insights into the development of a drug that prevents virus entry into host cells.

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