scholarly journals Mycobacterium tuberculosis senses host Interferon- gamma via the membrane protein MmpL10

2021 ◽  
Author(s):  
Mohamed Ahmed ◽  
Jared Stuart Mackenzie ◽  
Robert Krause ◽  
Barry Truebody ◽  
Liku Bekele Tezera ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Membrane Protein ◽  
Immune Defense ◽  
Mass Spectrometry Analysis ◽  
Resistant Bacteria ◽  
Bacterial Respiration ◽  
Human Pathogens ◽  
Spectrometry Analysis ◽  
Dose Dependent Increase ◽  
Dose Dependent

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens and remains a leading cause of death from infectious disease. Interferonγ (IFNγ) is a central regulator of the immune defense against Mtb. Several cytokines have been shown to increase virulence of other bacterial pathogens, leading us to investigate whether IFNγ has a direct effect on Mtb. We found that both recombinant and T cell derived IFNγ rapidly induced a dose dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was also observed in clinical strains, but not in the vaccine strain Bacillus Calmette Guerin (BCG), and did not occur for other cytokines tested, including TNFα. IFNγ binds to the cell surface of intact Mtb, but not BCG, whilst TNFα binds to neither. Mass spectrometry analysis identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner. Consistent with this, IFNγ binding and the OCR response was absent in a Mtb Δmmpl10 strain and restored by complementation of the mutant strain. RNA-sequencing of IFNγ exposed Mtb revealed a distinct transcriptional profile, including genes involved in virulence and cholesterol catabolism. Finally, exposure of Mtb cells to IFNγ resulted in sterilization of bacilli treated with isoniazid (INH), indicating clearance of phenotypically resistant bacteria that persist in the presence of INH alone. Our data suggest a novel mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies.

scholarly journals ENIGMATIC INDUCTION OF CYTOMIXIS IN ALLIUM CEPA ROOT MERISTEM BY AGLAIA EDULIS ROXB. LEAF EXTRACT AND ITS PHYTOCHEMICAL RATIONALE

2020 ◽  
pp. 168-171
Author(s):  
ARCHANA ELAMKULAM RAVINDRAN ◽  
JOHN ERNEST THOPPIL
Keyword(s):  
Allium Cepa ◽  
Leaf Extract ◽  
Root Meristem ◽  
Methanolic Extract ◽  
Synergistic Action ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Phytochemical Analysis ◽  
Spectrometry Analysis ◽  
Dose Dependent

Objective: The present study aims to analyze the potential of Aglaia edulis Roxb. leaf extract to induce cytological aberrations in Allium cepa root meristem and to determine the phytoconstituents in the extract. Methods: Cytotoxicity evaluation of the leaf methanolic extract was done using Allium cepa assay using various concentrations. Volatile phytoconstituents in the extract were determined using gas chromatography–mass spectrometry analysis. Results: Considerable number of cytomictic cells along with other aberrations was observed. The occurrence of cytomixis was found to be dose dependent where it ranged from 6.58±0.35 to 29.45±0.45. The percentage of cytomictic cells among the total aberrant cells was observed between 35.19±1.67 and 77.39±1.39. The phytochemical analysis of the plant extract revealed the presence of active secondary metabolites. Conclusion: The synergistic action of the active compounds might have triggered the phenomenon of cytomixis which, in turn, could be exploited for the production of polyploids.

scholarly journals Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1531-1540 ◽  
Author(s):  
Rahul Shrivastava ◽  
Bhakti Basu ◽  
Ashwini Godbole ◽  
M. K. Mathew ◽  
Shree K. Apte ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Organic Acids ◽  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Aromatic Compounds ◽  
Mass Spectrometry Analysis ◽  
Binding Studies ◽  
Spectrometry Analysis ◽  
Glucose Binding

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [14C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain’s ability to utilize aromatics and organic acids over glucose.

scholarly journals The Variability of Thymol and Carvacrol Contents Reveals the Level of Antibacterial Activity of the Essential Oils from Different Accessions of Oliveria decumbens

Antibiotics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 409
Author(s):  
Tahereh Khoshbakht ◽  
Akbar Karami ◽  
Aminallah Tahmasebi ◽  
Filippo Maggi
Keyword(s):  
Antibacterial Activity ◽  
Essential Oils ◽  
Positive Control ◽  
Mass Spectrometry Analysis ◽  
Human Pathogens ◽  
Clavibacter Michiganensis ◽  
Spectrometry Analysis ◽  
High Level ◽  
Shed Light ◽  
Chromatographic Mass

Oliveria decumbens (Apiaceae) is an aromatic herb traditionally employed in the Persian medicine for the treatment of infectious and gastrointestinal disorders. In the present study, we analyzed the chemical composition of essential oils obtained from different Iranian populations and evaluated their efficacy on a panel of human pathogens (Staphylococcus aureus and Escherichia coli), probiotic (Bacillus subtilis), and phytopathogens (Clavibacter michiganensis, Curtobacterium flaccumfaciens, Xanthomonas citri, and Agrobacterium tumefaciens). The gas chromatographic-mass spectrometry analysis put in evidence four main volatile constituents such as thymol (20.3–36.4%), carvacrol (18.8–33.1%), γ-terpinene (10.6–25.9%), and p-cymene (9.5–17.3%), though with significant variability from an essential oil to another. Notably, the oils from the populations sited in Nourabad Mamasani and Dehdasht showed the highest amount of the phenolic monoterpenes thymol (36.4 and 35.2%, respectively) and carvacrol (33.1 and 30.6%, respectively). The antibacterial activity of O. decumbens essential oils was assessed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods, showing high activity for the samples from Nourabad Mamasani and Dehdasht populations exhibiting high level of the above phenolics. The obtained MIC and MBC values (mg/ml) were in the ranges 0.0625–2 mg/ml and 1–16 mg/ml, respectively. Noteworthy, in some cases, the antibacterial activity of O. decumbens essential oils was higher than that of chloramphenicol used as positive control. The average MBCs displayed by the O. decumbens samples showed that C. flaccumfaciens had the highest sensitivity to the essential oils. Based on these results, our work shed light on selected O. decumbens populations deserving proper breeding and cultivation strategies in order to warrantee production of bioactive essential oils to be used at pharmaceutical and agricultural level to combat several pathogens.

scholarly journals Mycobacterium tuberculosis Thymidylyltransferase RmlA Is Negatively Regulated by Ser/Thr Protein Kinase PknB

2021 ◽  
Vol 12 ◽  
Author(s):  
Dehui Qu ◽  
Xiaohui Zhao ◽  
Yao Sun ◽  
Fan-Lin Wu ◽  
Sheng-Ce Tao
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Catalytic Triad ◽  
Mass Spectrometry Analysis ◽  
Wall Structure ◽  
Cell Wall Formation ◽  
Phenotypic Analysis ◽  
Spectrometry Analysis ◽  
Wall Formation

Ser/Thr phosphorylation by serine/threonine protein kinases (STPKs) plays significant roles in molecular regulation, which allows Mycobacteria to adapt their cell wall structure in response to the environment changes. Identifying direct targets of STPKs and determining their activities are therefore critical to revealing their function in Mycobacteria, for example, in cell wall formation and virulence. Herein, we reported that RmlA, a crucial L-rhamnose biosynthesis enzyme, is a substrate of STPK PknB in Mycobacterium tuberculosis (M. tuberculosis). Mass spectrometry analysis revealed that RmlA is phosphorylated at Thr-12, Thr-54, Thr-197, and Thr-12 is located close to the catalytic triad of RmlA. Biochemical and phenotypic analysis of two RmlA mutants, T12A/T12D, showed that their activities were reduced, and cell wall formation was negatively affected. Moreover, virulence of RmlA T12D mutant was attenuated in a macrophage model. Overall, these results provide the first evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mycobacteria, with significant implications for pathogenicity.

scholarly journals Proteomic Characterization of Antibiotic Resistance, and Production of Antimicrobial and Virulence Factors in Streptococcus Species Associated with Bovine Mastitis. Could Enzybiotics Represent Novel Therapeutic Agents Against These Pathogens?

Antibiotics ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 302 ◽  
Author(s):  
Ana G. Abril ◽  
Mónica Carrera ◽  
Karola Böhme ◽  
Jorge Barros-Velázquez ◽  
José-Luis R. Rama ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Research Work ◽  
Bovine Mastitis ◽  
Mass Spectrometry Analysis ◽  
Resistant Bacteria ◽  
Toxic Substances ◽  
Antibiotic Resistant ◽  
Spectrometry Analysis ◽  
Species Specific

Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC–ESI–MS/MS (liquid chromatography–electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strains.

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

2000 ◽  
Vol 278 (5) ◽  
pp. L1025-L1031 ◽  
Author(s):  
Sha Zhu ◽  
Khaled F. Basiouny ◽  
John P. Crow ◽  
Sadis Matalon
Keyword(s):  
Nitric Oxide ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Dose Dependent Fashion ◽  
Carbonyl Formation ◽  
Oxide Synthase ◽  
Dose Dependent

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO2. In the presence of CO2, lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion ofl-[U-14C]arginine tol-[U-14C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 ± 3 vs. 6 ± 1 (SE) nmol ⋅ 6.5 h−1 ⋅ 106AMs−1]. Western blotting studies of immunoprecipitated SP-A indicated that CO2 enhanced SP-A nitration by AMs and decreased carbonyl formation. CO2(0–1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr164 and Tyr166) in the absence of CO2 and three tyrosines (Tyr164, Tyr166, and Tyr161) in the presence of 1.2 mM CO2. These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO2 increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

scholarly journals Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1015
Author(s):  
Salvador Vazquez Reyes ◽  
Supriyo Ray ◽  
Javier Aguilera ◽  
Jianjun Sun
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lipid Membranes ◽  
Effect Of Temperature ◽  
In Vitro Assays ◽  
Vitro Model ◽  
First Time ◽  
Dose Dependent Increase ◽  
Dose Dependent

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.

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