In vitro and in silico assessment of anti-biofilm and anti-quorum sensing properties of 2,4-Di-tert butylphenol against Acinetobacter baumannii.

Introduction. Acinetobacter baumannii is a nosocomial pathogen with a high potential to cause food-borne infections. It is designated as a critical pathogen by the World Health Organization due to its multi-drug resistance and mortalities reported. Biofilm governs major virulence factors, which promotes drug resistance in A. baumannii. Thus, a compound with minimum selection pressure on the pathogen can be helpful to breach biofilm-related virulence.Hypothesis/Gap Statement. To identify anti-biofilm and anti-virulent metabolites from extracts of wild Mangifera indica (mango) brine pickle bacteria that diminishes pathogenesis and resistance of A. baumannii.Aim. This study reports anti-biofilm and anti-quorum sensing (QS) efficacy of secondary metabolites from bacterial isolates of fermented food origin.Method. Cell-free supernatants (CFS) of 13 bacterial isolates from fermented mango brine pickles were screened for their efficiency in inhibiting biofilm formation and GC-MS was used to identify its metabolites. Anti-biofilm metabolite was tested on early and mature biofilms, pellicle formation, extra polymeric substances (EPS), cellular adherence, motility and resistance of A. baumannii. Gene expression and in silico studies were also carried out to validate the compounds efficacy.Results. CFS of TMP6b identified as Bacillus vallismortis, inhibited biofilm production (83.02 %). Of these, major compound was identified as 2,4-Di-tert-butyl phenol (2,4-DBP). At sub-lethal concentrations, 2,4-DBP disrupted both early and mature biofilm formation. Treatment with 2,4-DBP destructed in situ biofilm formed on glass and plastic. In addition, key virulence traits like pellicle (77.5 %), surfactant (95.3 %), EPS production (3-fold) and cell adherence (65.55 %) reduced significantly. A. baumannii cells treated with 2,4-DBP showed enhanced sensitivity towards antibiotics, oxide radicals and blood cells. Expression of biofilm-concomitant virulence genes like csuA/B, pgaC, pgaA, bap, bfmR, katE and ompA along with QS genes abaI, abaR significantly decreased. The in silico studies further validated the higher binding affinity of 2,4-DBP to the AbaR protein than the cognate ligand molecule.Conclusion. To our knowledge, this is the first report to demonstrate 2,4- DBP has anti-pathogenic potential alone and with antibiotics by in vitro, and in silico studies against A. baumannii. It also indicates its potential use in therapeutics and bio-preservatives.

Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

Caveolin-1 scaffolding domain peptide regulates glucose metabolism in lung fibrosis.

Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Furthermore, the antifibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involving metabolic reprogramming in fLfs are unclear. We therefore analyzed lactate and succinate levels, as well as the expression of glycolytic enzymes and hypoxia inducible factor-1α (HIF-1α). Lactate and succinate levels, as well as the basal expression of glycolytic enzymes and HIF-1α, were increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes, and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or Ad-TGF-ß1-induced lung fibrosis with CSP/CSP7 reduced the expression of glycolytic enzymes and HIF-1α. Furthermore, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.

Caveolin-1-derived peptide limits development of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-ß1 (Ad-TGF-ß1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases.

Hydrocinnamic acid produced by Enterobacter xiangfangensis impairs AHL-based quorum sensing and biofilm formation in Pseudomonas aeruginosa.

Many of the Gram-negative bacteria regulate their virulence through an AHL-mediated quorum sensing (QS) mechanism. Disruption of this signaling mechanism might be a novel strategy to suppress bacterial virulence. In this report, foodborne bacterial isolates were tested for their QS-inhibitory properties using biosensor strain Chromobacterium violaceum CV026 and the extracted potential active components were evaluated for anti-QS and antibiofilm activity against pathogenic bacteria. The cell-free supernatant of Enterobacter xiangfangensis PUFSTI26 inhibited violacein production in the reporter strain and exhibited a significant reduction in extracellular virulence factors like biofilm formation, pyocyanin production, and motility of Pseudomonas aeruginosa. Characterization of the purified active component by gas chromatography-mass spectrometry (GC-MS) flaunted the resemblance of hydrocinnamic acid (HCA). Treatment of HCA exhibited pronounced attenuation of virulence factors. Further, the biofilm inhibitory activity was evidenced by means of confocal laser microscopy, that evidenced the repression of biofilm biomass. In addition, gene quantification analysis revealed that HCA repressed the expression of major QS-regulated genes. In silico studies showed that HCA competitively interacts with LasR receptor protein. These results clearly indicate the anti-virulence properties of HCA extracted from E. xiangfangensis of food origin. This is also the first report of the QS inhibitor activity of HCA.

p53 Expression in Lung Fibroblasts Is Linked to Mitigation of Fibrotic Lung Remodeling.

Idiopathic pulmonary fibrosis (IPF) is a debilitating, incurable, and life-threatening disease. A cardinal feature of the pathogenesis of IPF is excessive extracellular matrix deposition attributable to proliferation of activated fibrotic lung fibroblasts (fLfs). To assess the underlying mechanism, we analyzed the status of the tumor suppressor protein p53 in fLfs from the lungs of IPF patients or mice with bleomycin-induced established PF. We report that basal expression of p53 is markedly reduced in fLfs. Forced expression of caveolin-1 in fLfs increased basal p53 and reduced profibrogenic proteins, including collagen-1. Transduction of fLfs with adenovirus expressing p53 reduced expression of these proteins. Conversely, inhibition of baseline p53 in control lung fibroblasts from lung tissues increased profibrogenic protein expression. Lung transduction of adenovirus expressing p53 reduced bleomycin-induced PF in wild-type or caveolin-1-deficient mice. Furthermore, treatment of fLfs or fibrotic lung tissues with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored p53 and reduced profibrogenic proteins. Treatment of wild-type mice with i.p. CSP or CSP7 resolved bleomycin-induced PF. These peptides failed to resolve PF in inducible conditional knockout mice lacking p53 in fLfs, indicating the induction of baseline fLf p53 as the basis of the antifibrotic effects.

Regulation of acylated homoserine lactones (AHLs) in beef by spice marination.

Quorum sensing (QS) is a signaling mechanism used by bacteria to communicate each other through the release of auto-inducing signaling molecules. Despite the fact that bacteria regulate its phenotypes by QS mechanism, their potential role in meat spoilage is not yet elucidated. In the current study, beef samples were analyzed for its microbial association and for the presence of N-acyl-homoserine-lactone (AHLs) throughout the storage experiments. Isolates were screened for AHLs production and selected spices were screened for their quorum sensing inhibitory (QSI) activity. In addition, effect of spices on AHLs production of Y. enterocolitica was quantified through high performance thin layer chromatography (HP-TLC). Outcome showed that microbial association of beef mainly consists of lactic acid bacteria (LAB) and Enterobacteriaceae. Samples stored at both aerobic and modified atmospheric packaging (MAP) exhibited higher counts whereas; marinated samples stored at MAP exhibited the lowest. It was found that out of 35 isolates Y. enterocolitica induced reporter strain CV026 and its cell-free supernatant contained 26.36 nM/100 ml of AHLs when compared to standard. Among the tested spices, C. cyminum exhibited pronounced results by significantly reducing the AHLs concentration up to 47.75 %. Findings revealed the presence of quorum molecules (AHLs) in beef meat throughout the spoilage process and spices can acts as quorum quenchers to influence the spoilage rate by reducing AHLs production.

Cyanidin inhibits quorum signalling pathway of a food borne opportunistic pathogen.

Quorum sensing (QS) is the process of population dependent cell to cell communication used by bacteria to regulate their phenotypic characteristics. Key virulence factors that determine the bacterial pathogenicity and food spoilage were found to be regulated by QS mechanism. Hence, disrupting the QS signaling pathway could be an attractive strategy to manage food borne pathogens. In the current study, QS inhibitory activity of a naturally occurring anthocyanin-cyanidin and its anti-biofilm property were assessed against an opportunistic pathogen Klebsiella pneumoniae, using a bio-sensor strain. Further, QS inhibitory property of a naturally occurring anthocyanin cyanidin was further confirmed using in-silico techniques like molecular docking and molecular dynamics simulation studies. Cyanidin at sub-lethal dose significantly inhibited QS-dependent phenotypes like violacein production (73.96 %), biofilm formation (72.43 %), and exopolysaccharide production (68.65) in a concentration-dependent manner. Cyanidin enhanced the sensitivity of test pathogen to conventional antibiotics in a synergistic manner. Molecular docking analysis revealed that cyanidin binds more rigidly with LasR receptor protein than the signaling compound with a docking score of -9.13 Kcal/mol. Molecular dynamics simulation predicted that QS inhibitory activity occurs through the conformational changes between the receptor and cyanidin complex. Our results indicate that cyanidin, can be a potential QS based antibiofilm and antibacterial agent for food borne pathogens.

Quercetin Influences Quorum Sensing in Food Borne Bacteria: In-Vitro and In-Silico Evidence.

Quorum sensing (QS) plays a vital role in regulating the virulence factor of many food borne pathogens, which causes severe public health risk. Therefore, interrupting the QS signaling pathway may be an attractive strategy to combat microbial infections. In the current study QS inhibitory activity of quercetin and its anti-biofilm property was assessed against food-borne pathogens using a bio-sensor strain. In addition in-silico techniques like molecular docking and molecular dynamics simulation studies were applied to screen the quercetin's potentiality as QS inhibitor. Quercetin (80 µg/ml) showed the significant reduction in QS-dependent phenotypes like violacein production, biofilm formation, exopolysaccharide (EPS) production, motility and alginate production in a concentration-dependent manner. Synergistic activity of conventional antibiotics with quercetin enhanced the susceptibility of all tested pathogens. Furthermore, Molecular docking analysis revealed that quercetin binds more rigidly with LasR receptor protein than the signaling compound with docking score of -9.17 Kcal/mol. Molecular dynamics simulation predicted that QS inhibitory activity of quercetin occurs through the conformational changes between the receptor and quercetin complex. Above findings suggest that quercetin can act as a competitive inhibitor for signaling compound towards LasR receptor pathway and can serve as a novel QS-based antibacterial/anti-biofilm drug to manage food-borne pathogens.

Quorum quenching activity of Syzygium cumini (L.) Skeels and its anthocyanin malvidin against Klebsiella pneumoniae.

Many bacterial species use their intercellular signaling mechanism called quorum sensing (QS), which is found to be implicated in various factors including bacterial pathogenicity and food spoilage. Interrupting the bacterial communication is an attractive strategy to develop novel QS-based antibacterial drugs. Present study is aimed to investigate the quorum sensing inhibitory activity of Syzygium cumini and its anti-biofilm property against opportunistic pathogen using a biosensor strain Chromobacterium violaceum CV026. Ethanol extract of S. cumini was investigated for its anti-QS activity, and the possible active component was identified by docking with LasR receptor protein. Based on docking analysis, methanol extract was enriched for its total anthocyanin (STA) and its effect on QS regulated phenotypes was assessed. STA specifically inhibited the violacein production in C. violaceum; biofilm formation and EPS production in Klebsiella pneumoniae up to 82, 79.94 and 64.29% respectively. Synergistic activity of conventional antibiotics with STA enhanced the susceptibility of K. pneumoniae up to 58.45%. Molecular docking analysis of active components attributes the QSI activity of S. cumini to malvidin. Malvidin exhibited highest ligand binding with LasR receptor protein with docking score more than -7. Effect of malvidin to interrupt the QS regulated phenotypes was also assessed, and it was found to reduce the violacein production, biofilm formation and EPS production of K. pneumoniae in a concentration-dependent manner. These findings suggest that S. cumini can be used as novel QS-based antibacterial/anti-biofilm agent to manage food-borne pathogens and to increase food safety.