Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It’s antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis.

Nisin inhibits bacterial growth by generating pores in cell membrane and interrupting cell-wall biosynthesis through specific lipid II interaction. However, the role of the hinge region and C-terminus residues of the peptide in antibacterial action of nisin is largely unknown. Here, using molecular dynamics simulations and experimental approach, we report that at high concentration regimes of nisin, interaction with phospholipids may equally deform the bacterial cell membranes even under significantly varying amounts of lipid-II. Membrane thinning, destabilization and decrease in lipid density depend on the degree of oligomerization of nisin. Growth kinetics of Bacillus subtilis and Escherichia coli interestingly show recovery by extended lag phase under low concentrations of nisin treatment while high concentrations of nisin caused decrease in cell viability as recorded by striking reduction in membrane potential and surface area. The significant changes in the dipole potential and fluorescence anisotropy were observed in negatively charged membranes in the absence of lipid-II with increasing concentration of nisin. The identical correlation of cell viability, membrane potential dissipation and morphology with the concentration regime of nisin, in both Bacillus subtilis (lipid II rich) and Escherichia coli (lipid II impoverished), hints at a non-specific physical mechanism where degree of membrane deformation depends on degree of crowding and oligomerization of nisin.

Transient tunnels that assemble and disassemble to facilitate passage of unstable intermediates in enzymes containing multiple reaction centers are controlled by allosteric cues. Using the 140-kDa purine biosynthetic enzyme PurL as a model system and a combination of biochemical and x-ray crystallographic studies, we show that long-distance communication between ~25-Å distal active sites is initiated by an allosteric switch, residing in a conserved catalytic loop, adjacent to the synthetase active site. Further, combinatory experiments seeded from molecular dynamics simulations help to delineate transient states that bring out the central role of nonfunctional adaptor domains. We show that carefully orchestrated conformational changes, facilitated by interplay of dynamic interactions at the allosteric switch and adaptor-domain interface, control reactivity and concomitant formation of the ammonia tunnel. This study asserts that substrate channeling is modulated by allosteric hotspots that alter protein energy landscape, thereby allowing the protein to adopt transient conformations paramount to function.

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Parkia javanica is a well-known ethno-botanical plant of the north-east region of India. Ethnic communities of the region use several parts, including fruits, of this plant for the treatment of various ailments like diarrhoea, dysentery, cholera, food poisoning etc. In addition fruits are consumed by the local tribes of the north-east as food supplements. With this background we have performed chemical characterisation, and investigated the antimicrobial and antibiofilm potentiality of ethyl acetate fraction of Parkia javanica fruit extract (PJE) against model biofilm-causing microorganism Pseudomonas aeruginosa. PJE was initially prepared from fruit extract, and assayed by IR and UV spectroscopy and HPLC to confirm the presence of compounds. HPLC and NMR analysis reveals that PJE contains flavone compounds baicalein, quercetin and chrysin. PJE, baicalein, quercetin and chrysin were then tested for antimicrobial and antibiofilm activity against P. aeruginosa. PJE showed very significant antimicrobial activity against P. aeruginosa wherein the minimum inhibitory concentration was found at 180 mg mL À1. Interestingly, the antibiofilm study illustrates that minimum concentration of PJE (30 mg mL À1 ) exhibited maximum activity whereas maximum concentration of PJE (90 mg mL À1 ) exhibited minimum antibiofilm activity. It was also observed that compounds baicalein, quercetin and chrysin separately show lower to moderate antibiofilm activity in comparison to PJE. Molecular docking study indicates that baicalein, quercetin and chrysin have good binding affinity with bacterial quorum sensing and motility associated proteins. Furthermore, we have also observed that in comparison with higher concentration, lower concentration of PJE exhibited better attenuation in swarming motility, secretion of proteases and virulence factors like pyoverdin and pyocyanin. AFM study reveals that aggregates in PJE are smaller in size at low concentrations than at higher concentrations. Observations in the present study suggest that PJE as a whole shows higher antibiofilm activity at low concentration whereas individual compounds have comparatively lower antibiofilm activity. This validates the phytomedicinal significance of Parkia javanica against bacterial biofilms.

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