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1.
Microb Pathog ; 151: 104723, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33460747

RESUMO

Signal dependent microbial communication in Pseudomonas aeruginosa PAO1 is a typical phenomenon mediated by acyl homo-serine lactone molecules that helps in developing biofilm and enhance antibiotic resistance. Microbial sources provide insight to the hidden treasure of secondary metabolites, and these structurally diversified chemical motifs can be used as antimicrobial and anti-infective agents. In the present study, endophytic fungus, Colletotrichum gloeosporioides HM3 isolated from Carica papaya leaves was explored for anti-infective potential against P. aeruginosa PAO1. The crude extract of C. gloeosporioides HM3 displayed bacteriostatic effect on P. aeruginosa PAO1 growth at 750 µg/ml concentration. A significant decline was observed in the production of quorum sensing regulated virulence factors, i.e. 56.32%, 62.54%, and 66.67% of pyocyanin, chitinase, and elastase enzyme, respectively. A drastic reduction in pathogenic determinant behaviour after treatment with crude extract of C. gloeosporioides HM3 i.e. EPS, rhamnolipid, and HCN production was noted. Light microscopy and CLSM analysis revealed that fungal extract treatment has reduced bacterial ability to form dense biofilm architecture. In silico analysis demonstrated the binding efficiency of bioactive compound, 4-(2,3-dimethoxybenzylidene)-3-methyl-1-(4-nitrophenyl)-2-pyrazolin-5-one, which is equipotent to the natural ligand and displayed a docking score of -5.436 kcal/mol with QS transcriptional regulator (LasR). Whereas the compound Acetamide, n-[tetrahydro-3-(phenylmethyl) thieno [3,4-d]thiazol-2 (3 h)-ylidene]-, s,s-dioxide exhibits a docking score of -4.088 kcal/mol (LasR) and -1.868 kcal/mol (RhlR) with cognate receptor proteins. Henceforth, the research report suggests C. gloeosporioides HM3 derived metabolites could be considered as a potential inhibitors of QS regulated virulence factors and biofilm production in P. aeruginosa PAO1.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33109065

RESUMO

INTRODUCTION: Insect growth and metamorphosis are strictly dependent on the structural changes that occur in chitin containing tissues and organs. Chitin synthase catalyzes chitin polymerization by ß-(1, 4) glycosidic linkage of Nacetyl-D-glucosamine (GlcNAc) monomers; the major component of insect cuticles. Targeting this enzyme could be a promising strategy to control insect pests while avoiding adverse effects on coexisting populations. Nikkomycin Z and polyoxins are commercially available fungal inhibitors known to bind to the nucleotide-binding sites of insects and fungal chitin synthase. But the binding mode of chitin synthase has not been explored to date as its structure is not available yet. METHODS: To understand the structural features of the Chilo partellus chitin synthase enzyme (CpCHS), the threedimensional (3D) structure of the CpCHS catalytic domain was modeled using ROBETTA webserver. The obtained model was used to investigate the binding mode of its substrate, uridine diphosphate-N-acetyl-D-glucosamine (UDPGlcNAc), and inhibitors (nikkomycin Z and polyoxins) by molecular docking approach using Schrödinger Suite-Maestro v9.2. The docked complexes were further investigated for their interaction stability by performing molecular dynamics (MD) simulations using GROMACS v5.1.2. RESULTS: Our study highlighted the significance of various interactions made by CHS residues present in the Walker-B loop and donor-binding motifs with the substrate (UDP-GlcNAc), and GEDR motif with an acceptor (GlcNAc). Also, the interactions of the QRRRW motif while forming chitin polymer were explored. We observed that the inhibitors exhibited good binding affinity with these motifs, indicated by their docking and binding affinity scores. CONCLUSION: In vitro analysis suggested that nikkomycin Z showed higher inhibition of chitin synthase activity at a concentration of 2.5 µg.L-1 . Our study provided insights into the crucial interactions of chitin synthase while designing inhibitors against insect pests.

3.
J Biomol Struct Dyn ; : 1-14, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33000693

RESUMO

Chitin synthase (CHS) is one of the crucial enzymes that play an essential role in chitin synthesis during the molting process, and it is considered to be the specific target to control insect pests. Currently, there are no potent inhibitors available in the market, which specifically target this enzyme. Pyrimidine nucleoside peptide, nikkomycin Z, binds to nucleotide-binding sites of fungal and insect CHS. But, their mode of action is still fragmentary due to the lack of a 3Dstructure of CHS. Chilo partellus is a severe pest insect of major food crops such as maize and sorghum, in an attempt to target integument expressed cuticular CpCHS. The CpChsA cDNA was cloned, and subsequently, their developmental and tissue-specific expression was studied. The 3D structure of the CHS catalytic domain was modeled, after which natural compounds were screened using a virtual screening workflow and resulted in the identification of five hit molecules. Molecular dynamics simulations were performed to investigate the dynamics and interactions of hits with CpCHS. The obtained results revealed that the compounds kasugamycin, rutin and robinin could act as potent inhibitors of CpCHS. All three molecules were observed to significantly reduce the chitin production as validated using in vitro and in vivo studies. Thus, this study aims to provide a set of novel inhibitor molecules against CpCHS for controlling the pest population. Communicated by Ramaswamy H. Sarma.

4.
Indian J Microbiol ; 60(1): 70-77, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32089576

RESUMO

Endophytic fungi provide rich reservoir for novel antimicrobial compounds. An endophytic fungus, from Carica papaya plant identified as Phomopsis tersa, was investigated for attenuating the quorum sensing mediated pathogenicity of Pseudomonas aeruginosa PAO1. Crude extract of P. tersa was found to reduce the production of redox-active pigments-pyocyanin and pyoverdine in P. aeruginosa PAO1 by 92.46% and 71.55%, respectively at sub-MIC concentration of 900 µg/mL. In addition, the crude extract was also able to inhibit the expression of virulence factors involved in biofilm formation: exopolysaccharide (72.21%) and alginate (72.50%). Secretion of cell-lytic enzymes was also found to be reduced: chitinase by 79.73% and elastase by 74.30%. 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione identified from GC-MS analysis, displayed favorable molecular interactions with P. aeruginosa transcriptional regulators, LasR and RhlR with good docking scores of - 6.873 kJ/mol and - 6.257 kJ/mol, respectively. The study thus reveals the potential use of P. tersa for discovering drugs against infectious pathogens.

5.
J Microbiol Biotechnol ; 30(4): 571-582, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31986566

RESUMO

Quorum sensing (QS)-mediated infections cause severe diseases in human beings. The control of infectious diseases by inhibiting QS using antipathogenic drugs is a promising approach as antibiotics are proving inefficient in treating these diseases. Marine fungal (Pestalotiopsis sydowiana PPR) extract was found to possess effective antipathogenic characteristics. The minimum inhibitory concentration (MIC) of the fungal extract against test pathogen Pseudomonas aeruginosa PAO1 was 1,000 µg/ml. Sub-MIC concentrations (250 and 500 µg/ml) of fungal extract reduced QS-regulated virulence phenotypes such as the production of pyocyanin, chitinase, protease, elastase, and staphylolytic activity in P. aeruginosa PAO1 by 84.15%, 73.15%, 67.37%, 62.37%, and 33.65%, respectively. Moreover, it also reduced the production of exopolysaccharides (74.99%), rhamnolipids (68.01%), and alginate (54.98%), and inhibited the biofilm formation of the bacteria by 90.54%. In silico analysis revealed that the metabolite of P. sydowiana PPR binds to the bacterial QS receptor proteins (LasR and RhlR) similar to their respective natural signaling molecules. Cyclo(-Leu-Pro) (CLP) and 4-Hydroxyphenylacetamide (4-HPA) were identified as potent bioactive compounds among the metabolites of P. sydowiana PPR using in silico approaches. The MIC values of CLP and 4-HPA against P. aeruginosa PAO1 were determined as 250 and 125 µg/ml, respectively. All the antivirulence assays were conducted at sub-MIC concentrations of CLP (125 µg/ml) and 4-HPA (62.5 µg/ml), which resulted in marked reduction in all the investigated virulence factors. This was further supported by gene expression studies. The findings suggest that the metabolites of P. sydowiana PPR can be employed as promising QS inhibitors that target pathogenic bacteria.

6.
Microb Pathog ; 138: 103811, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31644930

RESUMO

The bacterial cell communication also termed as Quorum sensing (QS) system was involved in the expression of several virulence traits during Pseudomonas infection. The attenuating of this bacterial cell communication system is an attractive approach for the management of bacterial infections without the complication of resistance development. In this respect, the marine environment has gained significant attention due to its biodiversity and as a source of novel bioactive compounds. The present study aimed to screening effective QS inhibitors from marine associated fungal species for QS inhibitors. Twelve morphologically distinct fungal isolates were isolated from the wood of Avicennia marina from marine ecosystem. The anti-QS potential of fungal crude extract from was investigated in biosensor strain and test bacterium, Chromobacterium violaceum and Pseudomonas aeruginosa PAO1, respectively. Promising anti-QS activity was observed in the crude extract of one of the fungal isolate and identified by molecular characterization using internal transcribed spacer (ITS) region as Blastobotrys parvus PPR3. The anti-virulence and antibiofilm effects of ethyl acetate fractions from PPR3 against P. aeruginosa PAO1 were evaluated. The fungal metabolites responsible for the anti-QS activity of fungal crude extract was identified using gas chromatography-mass spectrometry (GC-MS). Furthermore, molecular docking studies were performed to understand the interaction of bioactive compounds with as receptors of P. aeruginosa PAO1. The crude extract of PPR3 showed reduction in different virulence traits of P. aeruginosa PAO1 such as production of pyocyanin, elastase, protease, chitinase, swimming and swarming motility, biofilm formation, exopolysaccharide production and alginate production at different sub-MIC concentrations. Interaction of bioactive metabolites with LasR and RhlR receptors of P. aeruginosa PAO1 was reported. The findings of the present study suggested that metabolites of B. parvus PPR3 interfere with QS system of P. aeruginosa PAO1 and alters the production of virulence factors.


Assuntos
Antibiose , Organismos Aquáticos , Biofilmes , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Saccharomycetales/fisiologia , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Filogenia , Percepção de Quorum/efeitos dos fármacos , Saccharomycetales/classificação , Saccharomycetales/isolamento & purificação , Relação Estrutura-Atividade , Virulência/efeitos dos fármacos
7.
Microb Pathog ; 131: 128-134, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959097

RESUMO

Quorum sensing (QS) is the cell density dependent communication network which coordinates the production of pathogenic determinants in majority of pathogenic bacteria. Pseudomonas aeruginosa causes hospital-acquired infections by virtue of its well-defined QS network. As the QS regulatory network in P. aeruginosa regulates the virulence determinants and antibiotic resistance, attenuating the QS system seems to be influential in developing next-generation anti-infective agents. In the current study, the QS attenuation potential of a flavonoid, mosloflavone was investigated against P. aeruginosa virulence and biofilm formation. Mosloflavone inhibited the pyocyanin production, LasB elastase and chitinase by 59.52 ±â€¯2.74, 35.90 ±â€¯4.34 and 61.18 ±â€¯5.52% respectively. The QS regulated biofilm formation and development was also reduced when supplemented with sub-MIC of mosloflavone. The gene expression studies of mosloflavone using RT-PCR depicted its ability to down-regulate the expression levels of QS regulated virulence genes such as lasI (60.64%), lasR (91.70%), rhlI (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%) and pelA (50.45%). The down-regulation of QS virulence phenotypes by mosloflavone could be attributed to its binding affinity with the QS regulatory proteins, LasR and RhlR by competitively inhibiting the binding of natural autoinducers as evidenced from simulation studies. Mosloflavone also exhibited promising potential in controlling bacterial infection in Caenorhabditis elegans model system, in vivo. The anti-biofilm and anti-QS potential of mosloflavone in the current study illustrated the candidature of mosloflavone as a promising biocide.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Quitinases/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Transativadores/genética , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética
8.
Microb Pathog ; 118: 177-189, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29571725

RESUMO

In recent years, Pseudomonas aeruginosa PAO1 emerged as the significant pathogenic microorganism in majority of the hospital-acquired infections due to its resistance to the conventional antibiotics by virtue of its highly organized quorum sensing and associated biofilm formation. In the present study, quorum sensing attenuation potential of Diaporthe phaseolorum SSP12 extract was investigated against P. aeruginosa PAO1 amply supported by molecular docking studies. D. phaseolorum SSP12 extract significantly inhibited the production of LasI/R mediated LasA protease, LasB elastase and chitinase with 66.52 ±â€¯5.41, 71.26 ±â€¯4.58 and 61.16 ±â€¯4.28% of inhibition respectively at a concentration of 750 µg mL-1. In addition, RhlI/R mediated production of pyocyanin, exopolysaccharides and rhamnolipids were also down-regulated by 74.71 ±â€¯3.97, 66.41 ±â€¯3.62 and 63.75 ±â€¯3.76% respectively on treatment with sub-MIC concentration of D. phaseolorum SSP12. The light, fluorescence and confocal laser scanning microscopic (CLSM) analysis confirmed the significant disruption in biofilm formation. The presence of bioactive constituents such as phenyl ethylalcohol, 2, 4-di-tert-butylphenol, fenaclon, 1, 4-phenylenediacetic acid, and benzyl hydrazine in D. phaseolorum SSP12 extract was evident from Gas chromatography-mass spectrophotometric (GC-MS) analysis. From the in silico molecular docking studies, fenaclon and 2, 4-di-tert-butylphenol competitively binds to QS receptors LasR and RhlR and alters the binding of its cognate ligands and modulates the expression of virulence phenotypes. The promising anti quorum sensing efficacy of D. phaseolorum SSP12 extract suggested new avenues for development of anti-infective drugs from fungal derived metabolites to counteract the problems associated with conventional antibiotic therapies.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Ascomicetos/química , Ascomicetos/metabolismo , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Quitinases/metabolismo , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligases/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Polissacarídeos Bacterianos/metabolismo , Piocianina/metabolismo , Percepção de Quorum/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Virulência/efeitos dos fármacos , Fatores de Virulência/metabolismo
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