NirA is an alternative nitrite reductase fromPseudomonas aeruginosawith potential as an anti-virulence target

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

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Ethnomedicinal uses, Phytochemistry and Pharmacological Aspects of the Genus Berberis Linn: A Comprehensive Review

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323999201102141206 ◽

2020 ◽

Vol 23 ◽

Author(s):

Roohi Mohi-ud-din ◽

Reyaz Hassan Mir ◽

Prince Ahad Mir ◽

Saeema Farooq ◽

Syed Naiem Raza ◽

...

Keyword(s):

Chemical Constituents ◽

Pharmacological Activities ◽

Traditional Use ◽

Comprehensive Review ◽

Wide Range ◽

Anti Cancer ◽

Comprehensive Survey ◽

Ethnomedicinal Uses

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.

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Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

Infection and Immunity ◽

10.1128/iai.00575-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4462-4473 ◽

Cited By ~ 53

Author(s):

Michelle A. Laskowski ◽

Barbara I. Kazmierczak

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutational Analysis ◽

Response Regulator ◽

Acute Infection ◽

Opportunistic Pathogen ◽

Sensor Kinase ◽

Chronic Infections ◽

Reciprocal Regulation ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

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In Vitro Synergism of Colistin and N-acetylcysteine against Stenotrophomonas maltophilia

Antibiotics ◽

10.3390/antibiotics8030101 ◽

2019 ◽

Vol 8 (3) ◽

pp. 101 ◽

Cited By ~ 2

Author(s):

Nagaia Ciacci ◽

Selene Boncompagni ◽

Felice Valzano ◽

Lisa Cariani ◽

Stefano Aliberti ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Respiratory Infections ◽

Topical Administration ◽

Opportunistic Pathogen ◽

Wide Range ◽

Tract Infections ◽

Human Infections ◽

Time Kill ◽

Dose Dependent

Stenotrophomonas maltophilia is an emerging global opportunistic pathogen, responsible for a wide range of human infections, including respiratory tract infections. Intrinsic multidrug resistance and propensity to form biofilms make S. maltophilia infections recalcitrant to treatment. Colistin is among the second-line options in case of difficult-to-treat S. maltophilia infections, with the advantage of being also administrable by nebulization. We investigated the potential synergism of colistin in combination with N-acetylcysteine (NAC) (a mucolytic agent with antioxidant and anti-inflammatory properties) against S. maltophilia grown in planktonic phase and biofilm. Eighteen S. maltophilia clinical isolates (comprising three isolates from cystic fibrosis (CF) and two trimethoprim-sulfamethoxazole (SXT)-resistant strains) were included. Checkerboard assays showed a synergism of colistin/NAC combinations against the strains with colistin Minimum Inhibitory Concentration (MIC) >2 µg/mL (n = 13), suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Nonetheless, time–kill assays revealed that NAC might potentiate colistin activity also in case of lower colistin MICs. A dose-dependent potentiation of colistin activity by NAC was also clearly observed against S. maltophilia biofilms, also at sub-MIC concentrations. Colistin/NAC combinations, at concentrations likely achievable by topical administration, might represent a valid option for the treatment of S. maltophilia respiratory infections and should be examined further.

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Specific and Global RNA Regulators in Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms22168632 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8632

Author(s):

Petra Pusic ◽

Elisabeth Sonnleitner ◽

Udo Bläsi

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Binding ◽

Gene Networks ◽

Target Genes ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Intrinsic Resistance ◽

Large Gene

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.

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A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

Journal of Bacteriology ◽

10.1128/jb.00805-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 6870-6881 ◽

Cited By ~ 25

Author(s):

Gregory T. Robertson ◽

Timothy B. Doyle ◽

Qun Du ◽

Leonard Duncan ◽

Khisimuzi E. Mdluli ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Opportunistic Pathogen ◽

Binding Pocket ◽

Cross Resistance ◽

Dependent Manner ◽

Intrinsic Resistance ◽

Outer Membrane Channel

ABSTRACT Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.

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Botanical, Traditional Use, Phytochemical, and Toxicological of Arisaematis rhizoma

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/9055574 ◽

2021 ◽

Vol 2021 ◽

pp. 1-25

Author(s):

Chun-yan Qi ◽

Jing Wang ◽

Xu Wu ◽

Su-rong He ◽

Qiao Zhang ◽

...

Keyword(s):

Hot Spot ◽

Chemical Constituents ◽

Pharmacological Action ◽

Human Beings ◽

Traditional Use ◽

Traditional Uses ◽

Wide Range ◽

Online Library

Aim of the Study. This paper summarizes the traditional uses, botany, chemical composition, pharmacology, pharmacodynamics, and toxicology of Arisaematis rhizoma (hereafter referred to as AR). In addition, the future development and research prospect of AR were discussed in detail. Although many traditional uses of AR have been confirmed, it is essential to study the relationship between its structure and function, clarify the mechanism of pharmacological action, and explore new clinical applications. Materials and Methods. This review is a collection of all possible studies on AR, published in scientific journals, papers, and books. Using the papers related to Arisaematis, such as ScienceDirect, Wiley Online Library, Springer Link, Web of Science, CNKI, and WanFang Database. In this paper, the traditional uses, botany, phytochemistry, pharmacology, and toxicology of AR were reviewed. Finally, the existing problems and research directions of the research on AR are discussed. Results. Ninety-eight chemical constituents were isolated from AR. AR has a wide range of pharmacological effects, such as the effects on the central nervous system and cardiovascular system. It also has anti-tumor, sedative, analgesic, anticonvulsant, anti-inflammatory, expectorant, antiarrhythmic, anticoagulant, and other effects. It is also considered an effective drug for in vitro and in vivo validation. Conclusions. AR is an excellent traditional medicinal plant in China. Pharmacological studies support the traditional use of AR and may verify the folk use of AR in the treatment of different diseases. The anti-tumor effect of AR has been widely concerned by scholars at home and abroad. It has become a hot spot in recent years and has made great contributions to the survival and development of human beings. Although it has a high value of comprehensive utilization, its development and utilization are far from enough. Therefore, the comprehensive development of AR is worthy of further analysis.

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HGG-23. IN VITRO AND IN VIVO PRECLINICAL DRUG SCREENING OF PROMISING THERAPEUTICS FOR DIFFUSE MIDLINE GLIOMA (DMG)

Neuro-Oncology ◽

10.1093/neuonc/noab090.087 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i21-i22

Author(s):

Chiara Cianciolo Cosentino ◽

Sandra Laternser ◽

Justyna M Przystal ◽

Sridevi Yadavilli ◽

Jie Zhang ◽

...

Keyword(s):

Acute Toxicity ◽

Tumor Biology ◽

In Vivo Studies ◽

Continuous Exposure ◽

Intrinsic Resistance ◽

Drug Candidates ◽

Zebrafish Larvae ◽

Wide Range

Abstract Introduction Diffuse midline gliomas (DMGs) are amongst the most unforgiving pediatric brain tumors, characterized by an intrinsic resistance to therapy. Despite major advances in understanding of tumor biology, the prognosis remains exceedingly poor, and treatment options are limited. New therapeutics are being evaluated at a fast rate by different laboratories. In order to prioritize effective drug candidates for DMG treatment, we comprehensively characterized a panel of promising therapeutic agents in in vitro and in different vivo systems. Methods We determined the sensitivity of primary DMG cell lines to a panel of small molecule inhibitors targeting known DMG targets and pathways. Dose response curves were generated for more than 20 different compounds and possible synergistic effects were investigated by SynergieFinder. In an effort to highlight potential toxicities and associated mechanisms at a large scale, we performed a preclinical toxicity evaluation in zebrafish larvae, with a slightly modified version of the official Fish Embryo Acute Toxicity (FET) test. Drug toxicity was tested by continuous exposure of zebrafish larvae to increasing concentrations of the different compounds. Survival curves, morphological analyses and behavioral tests were performed at a maximum tolerated dose (MTD). To confirm the findings obtained in zebrafish, we further performed in vivo studies in mice for promising candidates. Results Among the tested drugs in vitro we found 10 drugs showing promising dose- dependent reduction in cell viability with IC50 in nM to µM range. These were further evaluated for toxicity in zebrafish. The zebrafish larvae toxicities observations strongly correlated with the findings in murine in vivo studies, reinforcing the importance of zebrafish as an accurate investigative toxicology model to assess acute toxicity of molecules in preclinical studies. Conclusions By testing a wide range of drugs, targeting different pathways on DMG cells and in different in vivo systems we identified promising drug candidates for clinical management of children diagnosed with DMG.

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Combining amoxicillin and relebactam provides new therapeutic potential for Mycobacterium abscessus infection

10.1101/563189 ◽

2019 ◽

Author(s):

Rose C. Lopeman ◽

James Harrison ◽

Maya Desai ◽

Peter Lambert ◽

Jonathan A. G. Cox

Keyword(s):

De Novo ◽

Competitive Inhibition ◽

Therapeutic Potential ◽

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Therapeutic Interventions ◽

Intrinsic Resistance ◽

Lactam Antibiotic ◽

Significant Step

AbstractInfections caused by the opportunistic pathogen Mycobacterium abscessus are increasing in prevalence within the cystic fibrosis population. Our limited understanding of this ubiquitous environmental microorganism matched with its intrinsic resistance to most classes of antibiotic, including β-lactams, has left us with an urgent demand for new, more effective therapeutic interventions. De novo antimicrobial drug discovery is a lengthy process and so we have taken the approach of repurposing known antibiotics in order to provide a rapidly implementable solution to a current problem. Here we report a significant step forward in treatment potential for M. abscessus infection by sensitising the organism to the broad spectrum β-lactam antibiotic, amoxicillin, using the non-competitive β-lactamase inhibitor, relebactam. We demonstrate by disk diffusion and broth microdilution assay that this combination works synergistically to inhibit M. abscessus. We also demonstrate the direct non-competitive inhibition of the M. abscessus β-lactamase, BlaMab using a novel thin-layer chromatography-based assay for β-lactamase inhibition, which is subsequently kinetically validated by spectrophotometric assay using the nitrocefin reporter assay. Finally, we demonstrate the in vitro efficacy of this combination against a collection of M. abscessus clinical isolates, demonstrating the significant therapeutic potential of the amoxicillin and relebactam combination.

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Potential Medicinal Plants for the Treatment of Dengue Fever and Severe Acute Respiratory Syndrome-Coronavirus

Biomolecules ◽

10.3390/biom11010042 ◽

2020 ◽

Vol 11 (1) ◽

pp. 42

Author(s):

Mohammed S. M. Saleh ◽

Yusof Kamisah

Keyword(s):

Medicinal Plants ◽

Severe Acute Respiratory Syndrome ◽

Denv Infection ◽

In Vitro Studies ◽

In Vivo Studies ◽

Traditional Use ◽

Denv Serotypes ◽

Wide Range

While dengue virus (DENV) infection imposes a serious challenge to the survival of humans worldwide, severe acute respiratory syndrome-coronavirus (SARS-CoV) remains the most devastating pandemic in human history. A significant number of studies have shown that plant-derived substances could serve as potential candidates for the development of safe and efficacious remedies for combating these diseases. Different scientific databases were used to source for literature on plants used against these infections. Thirty-five studies described the traditional use of 25 species from 20 families for treating DENV infection with Carica papaya and Euphorbia hirta were the most widely used across different regions. 13 in vivo studies, 32 in vitro studies, and eight clinical studies were conducted on 30 species from 25 families against different DENV serotypes, while plants from 13 families were reported to inhibit different forms of SARS-CoV, all of which were investigated through in vitro studies. Phytoconstituents belonging to various chemical classes were identified to show a wide range of antiviral activity against these infections. Extensive studies on the potentials of medicinal plants are needed to confirm their efficacy. This paper reveals the capabilities of medicinal plants and their phytochemicals in inhibiting DENV and SARS-CoV infections.

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