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Design of a gold nanoprobe for the detection of Pseudomonas aeruginosa elastase gene (lasB).

ABSTRACT: Nosocomial infections are one of the major health problems that increase mortality. Pseudomonas aeruginosa is one of the important causes of nosocomial infections. This bacterium has a gene called the lasB gene, the product of which is a zinc-dependent metalloprotease. This gene plays a central role in the pathogenesis of Pseudomonas spp. This pathogen is highly toxic and destroys tissues and also has a moderating effect on the immune system; on the other hand, it initiates the intracellular pathway of biofilm growth. Although there are methods such as molecular methods for identifying the lasB gene, due to the high cost and the need for specialized personnel, it is necessary to replace them with an appropriate method. In this study, a gold nanoparticle-based DNA diagnostic sensor sensitive to the aggregation states of gold nanoparticles was used to identify amplified and non-amplified lasB genes. The results of the experiment were evaluated both visually and spectrally. The minimum detection value of this method was 10 ng of the amplified lasB gene and 50 ng of the non-amplified lasB gene. This method is very fast, simple, easy and low cost.

SUBMITTER: Farhangi A

PROVIDER: S-EPMC9051652 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Design of a gold nanoprobe for the detection of Pseudomonas aeruginosa elastase gene (lasB).

Farhangi Alireza A Peymani Amir A Ahmadpour-Yazdi Hossien H

RSC advances 20200317 20

Nosocomial infections are one of the major health problems that increase mortality. Pseudomonas aeruginosa is one of the important causes of nosocomial infections. This bacterium has a gene called the lasB gene, the product of which is a zinc-dependent metalloprotease. This gene plays a central role in the pathogenesis of Pseudomonas spp. This pathogen is highly toxic and destroys tissues and also has a moderating effect on the immune system; on the other hand, it initiates the int ...[more]

PMID: 35496606

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Project description:With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 μg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections. KEY POINTS: • Quercetin diminished the content and activity of LasB elastase of P. aeruginosa. • Quercetin inhibited the QS system activity of P. aeruginosa. • Quercetin acted on LasB based on the QS system.

Project description:Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K i = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa.

Dataset Information

Design of a gold nanoprobe for the detection of Pseudomonas aeruginosa elastase gene (lasB).

Publications

Design of a gold nanoprobe for the detection of <i>Pseudomonas aeruginosa</i> elastase gene (lasB).

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