scholarly journals Targeting Streptococcus pneumoniae UDP-glucose pyrophosphorylase (UGPase): in vitro validation of a putative inhibitor

2020 ◽  
Vol 14 (1) ◽  
pp. 26-33
Author(s):  
Monica Sharma ◽  
Swati Sharma ◽  
Pallab Ray ◽  
Anuradha Chakraborti
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Inhibitory Concentration ◽  
A549 Cells ◽  
Host Cells ◽  
Genome Plasticity ◽  
Leloir Pathway ◽  
Tnf Α ◽  
Respiratory Epithelial

Background: Genome plasticity of Streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide based vaccines. Therefore targets independent of capsular types are sought to control the pneumococcal pathogenicity. UcrDP-glucose pyrophosphorylase (UGPase) is one such desired candidate being responsible for the synthesis of UDP-glucose, a sugar-precursor in capsular biosynthesis and metabolic Leloir pathway. Being crucial to pneumococcal pathobiology, the effect of UGPase inhibition on virulence was evaluated in vitro. Methods: A putative inhibitor (UDP) was evaluated for effective inhibitory concentration in S. pneumoniae and A549 cells, its efficacy and toxicity. Effect of UDP on adherence and phagocytosis was measured in human respiratory epithelial (A549 and HEp-2) and macrophage (THP1 and J774.A.1) cell lines respectively. Results: A differential effective inhibitory concentration of UDP for UGPase inhibition was observed in S. pneumoniae and A549 cells i.e. 5 µM and 100 µM respectively. UDP treatments lowered percent cytotoxicity in pneumococcal infected monolayers and didn't exert adverse effects on viabilities. S. pneumoniae adherence to host cells was decreased significantly with UDP treatments. UDP induced the secretion of IL-1β, TNF-α, IL-6, and IL-8 and increased pneumococcal phagocytosis. Conclusion: Our study shows UDP mediated decrease in the virulence of S. pneumoniae and demonstrates UDP as an effective inhibitor of pneumococcal UGPase.

Elevated plasma levels of soluble TNF receptors are associated with morbidity and mortality in patients with acute lung injury

2005 ◽  
Vol 288 (3) ◽  
pp. L426-L431 ◽  
Author(s):  
Polly E. Parsons ◽  
Michael A. Matthay ◽  
Lorraine B. Ware ◽  
Mark D. Eisner
Keyword(s):  
Lung Injury ◽  
Plasma Levels ◽  
A549 Cells ◽  
Multicenter Trial ◽  
Morbidity And Mortality ◽  
Lung Protective Ventilation ◽  
Single Center ◽  
Lung Protection ◽  
Tnf Α

Ventilator-induced lung injury (VILI) is an inflammatory process that can be attenuated by lung protective ventilation strategies. Our objectives to further investigate the pathogenesis of ALI and VILI and the mechanism of lung protection in these syndromes were: 1) to determine if plasma measurements of soluble TNF receptor I (sTNFRI) and II (sTNFRII) would predict the development of ALI and mortality in a small single center trial; 2) to test the predictive value of these markers and of TNF-α in a larger, broader group of patients with ALI; 3) to test the hypothesis that low tidal volume ventilation (LTVV) would be associated with a decrease in plasma levels of TNF-α, sTNFRI, and sTNFRII. In the single center study, sTNFRI and II levels were higher in patients at risk for and with ALI, but they did not predict the development of the syndrome. In the multicenter trial sTNFRI and II were strongly associated with mortality (OR 5.76/1 log10 increment in receptor level; 95% CI 2.63–12.6 and OR 2.58; 95% CI 1.05–6.31, respectively) and morbidity measured as fewer nonpulmonary organ failure-free and ventilator-free days. The LTVV strategy was associated with an attenuation of plasma sTNFRI levels. In vitro, stimulated A549 cells release sTNFRI but not sTNRFII. In conclusion, plasma levels of sTNFRI and II can serve as biomarkers for morbidity and mortality in patients with ALI. Furthermore, LTVV is associated with a specific decrease in sTNFRI levels. This suggests that one beneficial effect of LTVV may be to attenuate alveolar epithelial injury.

scholarly journals Autoinducer 2 Signaling via the Phosphotransferase FruA Drives Galactose Utilization by Streptococcus pneumoniae , Resulting in Hypervirulence

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Claudia Trappetti ◽  
Lauren J. McAllister ◽  
Austen Chen ◽  
Hui Wang ◽  
Adrienne W. Paton ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Capsular Polysaccharide ◽  
The Body ◽  
Gram Negative Bacteria ◽  
Phosphotransferase System ◽  
Gram Positive ◽  
Gram Negative ◽  
Autoinducer 2 ◽  
Leloir Pathway

ABSTRACT Communication between bacterial cells is crucial for the coordination of diverse cellular processes that facilitate environmental adaptation and, in the case of pathogenic species, virulence. This is achieved by the secretion and detection of small signaling molecules called autoinducers, a process termed quorum sensing. To date, the only signaling molecule recognized by both Gram-positive and Gram-negative bacteria is autoinducer 2 (AI-2), synthesized by the metabolic enzyme LuxS ( S -ribosylhomocysteine lyase) as a by-product of the activated methyl cycle. Homologues of LuxS are ubiquitous in bacteria, suggesting a key role in interspecies, as well as intraspecies, communication. Gram-negative bacteria sense and respond to AI-2 via the Lsr ABC transporter system or by the LuxP/LuxQ phosphorelay system. However, homologues of these systems are absent from Gram-positive bacteria and the AI-2 receptor is unknown. Here we show that in the major human pathogen Streptococcus pneumoniae , sensing of exogenous AI-2 is dependent on FruA, a fructose-specific phosphoenolpyruvate-phosphotransferase system that is highly conserved in Gram-positive pathogens. Importantly, AI-2 signaling via FruA enables the bacterium to utilize galactose as a carbon source and upregulates the Leloir pathway, thereby leading to increased production of capsular polysaccharide and a hypervirulent phenotype. IMPORTANCE S. pneumoniae is a Gram-positive bacterium frequently carried asymptomatically in the human nasopharynx. However, in a proportion of cases, it can spread to other sites of the body, causing life-threatening diseases that translate into massive global morbidity and mortality. Our data show that AI-2 signaling via FruA promotes the transition of the pneumococcus from colonization to invasion by facilitating the utilization of galactose, the principal sugar available in the upper respiratory tract. AI-2-mediated upregulation of Leloir pathway enzymes results in increased production of capsular polysaccharide and hypervirulence in a murine intranasal challenge model. This identifies the highly conserved FruA phosphotransferase system as a target for new antimicrobials based on the disruption of this generic quorum-sensing system.

scholarly journals AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Rafael Ayerbe-Algaba ◽  
Nuria Bayó ◽  
Ester Verdú ◽  
Raquel Parra-Millán ◽  
Jesús Seco ◽  
...  
Keyword(s):  
Protein A ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Gram Negative ◽  
E Coli ◽  
Cyclic Hexapeptide ◽  
Diphenyltetrazolium Bromide ◽  
Peptide Derivatives

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.

scholarly journals THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

2018 ◽  
pp. 26-31
Author(s):  
I. V. Yakovleva ◽  
E. A. Kurbatova ◽  
E. A. Akhmatova ◽  
E. V. Sukhova ◽  
D. V. Yashunsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Solid Phase ◽  
Synthetic Analogue ◽  
Immunochemical Characterization ◽  
Carbohydrate Epitopes ◽  
First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

scholarly journals Synthesis and Antimicrobial Resistant Modulatory Activity of 2,4-Dinitrophenylhydrazone Derivatives as Agents against Some ESKAPE Human Pathogens

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Alberta Ade ◽  
Cedric D. K. Amengor ◽  
Abena Brobbey ◽  
Isaac Ayensu ◽  
Benjamin K. Harley ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Klebsiella Pneumoniae ◽  
Inhibitory Concentration ◽  
Fungal Species ◽  
Human Pathogens ◽  
Visible Spectroscopy ◽  
Active Compounds ◽  
Gram Negative ◽  
Uv Visible

A library of six novel phenylhydrazones were synthesized and evaluated for their in vitro antimicrobial and resistance modulating activity against a panel of Gram-positive, Gram-negative, and fungal species. The compounds were produced in good yields of 60–92% w/w and characterized using melting point, UV-visible spectroscopy, infrared, and nuclear magnetic resonance (1H, 13C, and DEPT-Q) techniques. Mass spectroscopy was used to confirm the identity of one of the most active compounds, 5 [SA5]. The phenylhydrazones showed activity against all the six selected microorganisms with minimum inhibitory concentration (MIC) values of the most active compounds, 1 [BP1] and 5 [SA5], at 138 µM (Klebsiella pneumoniae) and 165 µM (Streptococcus pneumoniae), respectively. Compound 1 [BP1] further demonstrated a high resistance modulatory activity at 1.078 µM against Streptococcus pneumoniae and Klebsiella pneumoniae.

scholarly journals Dendritic Cells Pulsed with Intact Streptococcus pneumoniae Elicit both Protein- and Polysaccharide-specific Immunoglobulin Isotype Responses In Vivo through Distinct Mechanisms

2001 ◽  
Vol 195 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Jesus Colino ◽  
Yi Shen ◽  
Clifford M. Snapper
Keyword(s):  
Dendritic Cells ◽  
Streptococcus Pneumoniae ◽  
Myeloid Dendritic Cells ◽  
Bacterial Proteins ◽  
Histocompatibility Complex ◽  
Tnf Α ◽  
Necrosis Factor ◽  
Igg1 Isotype

Immature bone marrow–derived myeloid dendritic cells (BMDCs) are induced to undergo phenotypic maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-10 when pulsed in vitro with intact Streptococcus pneumoniae. After transfer to naive mice, pulsed BMDCs induce immunoglobulin (Ig) isotype responses specific for both protein and polysaccharide pneumococcal antigens, having in common the requirement for viable BMDCs, T cells, and B7-dependent costimulation in the recipient mice. Whereas primary Ig isotype responses to bacterial proteins uniformly require BMDC expression of major histocompatibility complex class II, CD40, and B7, and the secretion of IL-6, but not IL-12, similar requirements for antipolysaccharide Ig responses were only observed for the IgG1 isotype.

scholarly journals Borrelia burgdorferiSpirochetes Induce Mast Cell Activation and Cytokine Release

1999 ◽  
Vol 67 (3) ◽  
pp. 1107-1115 ◽  
Author(s):  
Jeffrey Talkington ◽  
Steven P. Nickell
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Activation ◽  
Cell Types ◽  
Host Cells ◽  
Mast Cell Activation ◽  
Cytokine Release ◽  
Necrosis Factor Alpha ◽  
Tnf Α

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells.

Short-term modulation of interleukin-1β signaling by hyperoxia: uncoupling of IκB kinase activation and NF-κB-dependent gene expression

2004 ◽  
Vol 286 (3) ◽  
pp. L554-L562 ◽  
Author(s):  
Kelli Odoms ◽  
Thomas P. Shanley ◽  
Hector R. Wong
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Dna Binding ◽  
Nuclear Translocation ◽  
A549 Cells ◽  
Concomitant Treatment ◽  
Simultaneous Treatment ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Respiratory Epithelial

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-α (TNF-α)-mediated activation of NF-κB and NF-κB-dependent gene expression by a mechanism involving increased activation of IκB kinase (IKK). Because the signal transduction mechanisms induced by IL-1β are distinct to that of TNF-α, herein we sought to determine whether hyperoxia modulates IL-1β-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1β caused increased activation of IKK, prolonged the degradation of IκBα, and prolonged the nuclear translocation and DNA binding of NF-κB compared with cells treated with IL-1β alone in room air. Hyperoxia did not affect IL-1β-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-β alone. In contrast to the effects on the IKK/IκBα/NF-κB pathway, simultaneous treatment with hyperoxia and IL-1β did not augment NF-κB-dependent gene expression compared with treatment with IL-1β alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1β caused hyperphosphorlyation of the NF-κB p65 subunit compared with treatment with IL-1β alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1β augments activation of IKK, prolongs degradation of IκBα, and prolongs nuclear translocation and DNA binding of NF-κB. This activation, however, is not coupled to increased expression of NF-κB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.

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