Padmani Sandhu

Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin

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.

The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective.

The multidrug resistance has emerged as a major problem in the treatment of many of the infectious diseases. Tuberculosis (TB) is one of such disease caused by Mycobacterium tuberculosis. There is short term chemotherapy to treat the infection, but the main hurdle is the development of the resistance to antibiotics. This resistance is primarily due to the impermeable mycolic acid rich cell wall of the bacteria and other factors such as efflux of antibiotics from the bacterial cell. The MmpL (Mycobacterial Membrane Protein Large) proteins of mycobacteria are involved in the lipid transport and antibiotic efflux as indicated by the preliminary reports. We present here, comprehensive comparative sequence and structural analysis, which revealed topological signatures shared by the MmpL proteins and RND (Resistance Nodulation Division) multidrug efflux transporters. This provides evidence in support of the notion that they belong to the extended RND permeases superfamily. In silico modelled tertiary structures are in homology with an integral membrane component present in all of the RND efflux pumps. We document internal gene duplication and gene splitting events happened in the MmpL genes, which further elucidate the molecular functions of these putative transporters in an evolutionary perspective.

The drug binding sites and transport mechanism of the RND pumps from Mycobacterium tuberculosis: Insights from molecular dynamics simulations.

RND permease superfamily drug efflux pumps are involved in multidrug transport and are attractive to study them for therapeutic purpose. In previous work we have classified 14 members of MmpL proteins belong to RND superfamily from Mycobacterium tuberculosis (Mtb) within its families [Sandhu P. and Akhter Y., 2015. Int. J. Med. Microbiol., 305:413-423]. In this study, structures of these proteins are homology modelled. The drug binding sites and channels are identified using local micro-stereochemistry and charge densities. Potential transport mechanism based on differential structural behaviour in the absence and on the binding of drug molecules is explained using the molecular dynamics simulation results. Our studies show two potential drug binding sites positioned at opposite ends of the transport tunnel leading from cytoplasmic to the periplasmic space across MmpL5 trimer. The drug binding have effects on the structural conformation of the protein leading to molecular-scale peristaltic movements. The free binding energy calculations reveal that the subsequent binding events are interdependent and may have implications on transport mechanism. Two drug binding sites and a continuous channel in the RND pump have been reported. The proposed ligand binding mechanism shows peristaltic movements in the channel leading to the drug efflux. This study would be helpful in understanding the molecular basis of drugs resistance in Mtb.

Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture

Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.

Orchestration of membrane receptor signaling by membrane lipids

Receptors on cell membrane bind to their respective ligands and transduce intracellular signals resulting in variety of effector functions. Membrane lipid composition determines the receptor signaling behavior, as the receptors assume different conformation to suit the biochemical milieu in its immediate vicinity in the membrane. Accordingly, these accommodate different signaling intermediates that dictate the course of intracellular signaling and the resulting effectors functions. In this review we provide an overview of how membrane lipids modulate membrane-properties, membrane-receptor functions and their significance in the host-pathogen interaction.

Lipid-II Independent Antimicrobial Mechanism of Nisin Depends On Its Crowding And Degree Of Oligomerization

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.

Proteome scale census of major facilitator superfamily transporters in Trichoderma reesei using protein sequence and structure based classification enhanced ranking

Trichoderma spp. have been acknowledged as potent bio-control agents against microbial pathogens and also as plant growth promoters. Various secondary metabolites are attributed for these beneficial activities. Major facilitator superfamily (MFS) includes the large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites. We have carried out a proteome-wide identification of MFS transporters using protein sequence and structure based hierarchical method in Trichoderma reesei. 448 proteins out of 9115 were detected to carry transmembrane helices. MFS specific intragenic gene duplication and its context with transport function have been presented. Finally, using homology based techniques, domains and motifs of MFS families have been identified and utilized to classify them. From query dataset of 448 transmembrane proteins, 148 proteins are identified as potential MFS transporters. Sugar porter, drug: H(+) antiporter-1, monocarboxylate porter and anion: cation symporter emerged as major MFS families with 51, 35, 17 and 11 members respectively. Representative protein tertiary structures of these families are homology modeled for structure-function analysis. This study may help to understand the molecular basis of secretion and transport of agriculturally valuable secondary metabolites produced by these bio-control fungal agents which may be exploited in future for enhancing its biotechnological applications in eco-friendly sustainable development.

Antileishmanial and immunomodulatory activities of lupeol, a triterpene compound isolated from Sterculia villosa

Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis, caused by the protozoan parasite Leishmania donovani. Nowadays there is a growing interest in the therapeutic use of natural products to treat parasitic diseases. Sterculia villosa is an ethnomedicinally important plant. A triterpenoid was isolated from this plant and was screened for its antileishmanial and immunomodulatory activities in vitro and in vivo. Biochemical colour test and spectroscopic data confirmed that the isolated pure compound was lupeol. Lupeol exhibited significant antileishmanial activity, with IC50 values of 65 ± 0.41 µg/mL and 15 ± 0.45 µg/mL against promastigote and amastigote forms, respectively. Lupeol caused maximum cytoplasmic membrane damage of L. donovani promastigote at its IC50 dose. It is well known that during infection the Leishmania parasite exerts its pathogenicity in the host by suppressing nitric oxide (NO) production and inhibiting pro-inflammatory responses. It was observed that lupeol induces NO generation in L. donovani-infected macrophages, followed by upregulation of pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Lupeol was also found to reduce the hepatic and splenic parasite burden through upregulation of the pro-inflammatory response in L. donovani-infected BALB/c mice. Strong binding affinity of lupeol was observed for four major potential drug targets, namely pteridine reductase 1, adenine phosphoribosyltransferase, lipophosphoglycan biosynthetic protein and glycoprotein 63 of L. donovani, which also supported its antileishmanial and immunomodulatory activities. Therefore, the present study highlights the antileishmanial and immunomodulatory activities of lupeol in an in vitro and in vivo model of VL.

Antibiofilm activity of Parkia javanica against Pseudomonas aeruginosa: a study with fruit extract

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 μg mL−1. Interestingly, the antibiofilm study illustrates that minimum concentration of PJE (30 μg mL−1) exhibited maximum activity whereas maximum concentration of PJE (90 μg mL−1) 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.

Siderophore transport by MmpL5-MmpS5 protein complex in Mycobacterium tuberculosis

Iron is an essential metal ion required for the various physiological activities of bacteria. The pathogenic bacteria remain dependent on the host cell for their iron requirements and evolved with specialized scavenging machinery in the form of iron chelating siderophores. Mycobacterium tuberculosis has two types of siderophore molecules, mycobactin and carboxymycobactin. These are synthesized inside bacterial cells and need to be transported outside by specialized membrane associated proteins. MmpL5-MmpS5 (mycobacterial membrane protein large5-mycobacterial membrane protein small5) complex has been linked to the export of non-ferrated siderophores to extracellular environment but the precise molecular mechanism involved was largely unknown. We have investigated the association of MmpL5 with mycobactin synthesis and transport associated proteins using system wide protein-protein interaction network. Insights of mycobactin transport mechanism by MmpL5-MmpS5 complex was explored using docking and molecular dynamics simulations. The MmpL5 has association with many proteins with reported roles in iron acquisition or mycobactin biosynthesis. The molecular dynamics simulation analysis after mycobactin docking into MmpL5 binding pockets showed that at cytoplasmic binding site, mycobactin could move towards the central channel of efflux pump and at periplasmic binding site towards the periplasm. MmpL5 was observed to carry out uptake of mycobactin from the cytoplasm and its release into the periplasmic space and MmpS5 was found to facilitate the periplasmic release of mycobactin and enhancement in the transport function of MmpL5. The mycobactin export is an attractive target for drug discovery and it may be carried out by inhibiting the MmpL5 proteins transport function.