Toxicity and immunomodulatory efficacy of biosynthesized silver myconanosomes on pathogenic microbes and macrophage cells

<p>Silver myconanosomes prepared from <i>Alternaria brassicae</i> may exhibit potential antimicrobial and immunomodulatory activity due to their inimitable character. The prepared myconanosomes were characterized by using differential light scattering, zeta potential, UV–visible spectroscopy and transmission electron microscopic analyses. Mycologically produced AgNPs were found as spherical and irregular shaped measuring size range between 55.4 and 70.23 nm. The antimicrobicidal activity of these AgNPs against pathogenic microbes was evaluated by agar well diffusion method. Results showed that AgNPs inhibit the growth of various bacteria and fungi, which may be due to the disruption of cell membranes, leakage of cytoplasm and DNA degradation. Cytotoxicity analysis of AgNPs on cell lines revealed its dose dependent effect. Moreover, significant increase of intracellular reactive oxygen species was characterized in AgNPs treated cells after 4 h of incubation. Thus, AgNPs may have a significant advantage over conventional antibiotics as microorganisms are acquiring resistance against the broad range of available antibiotics.</p

<i>Wolbachia</i> Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

<div><p>Objectives</p><p><i>Wolbachia</i>, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains.</p><p>Methodology/Principal Findings</p><p>Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ββ′σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ββ′σ subunits of polymerase.</p><p>Conclusions/Significance</p><p>To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this α-proteobacteria and in deciphering the role of Wol GreA in filarial development.</p></div

Prediction of 3D structural model and validation of Wol GreA.

<p>(A) A computationally derived structural model of Wol GreA. The bioinformatics tool MODELLER9v10 was used to create a 3D ribbon model of Wol GreA that contained NTD (cyan) with RNA polymerase (RNAP) nucleolytic activity and CTD (red) possessing RNAP binding activity. (B) Structural superimposition of Wol GreA (red) with <i>E. coli</i> GreA (green) having PDB code1GRJ signifies an excellent template exhibiting 46% identity, 68% similarity, and 0.674 Å root mean square deviation (RMSD). (C) Ramachandran plot of Wol GreA constructed by PROCHECK revealed 89.8% a.a (red) residues of Wol GreA in most favored regions while 9.9% a.a (yellow) in additionally allowed regions and 0.3% a.a (pale yellow) in generously allowed regions, 0% a.a (white) residues were in disallowed regions. (D) Energy profile of Wol GreA was predicted by ProSA web server.</p

<i>In Silico</i> docking of Wol GreA with Wol RNAP subunits.

<p>For protein–protein docking the HEX program based on a rigid body protein docking algorithm that explicitly determines the steric shape, electrostatic potential, and charge density of the protein as expansions of spherical polar Fast Fourier Transformation (FFT) basis functions was exploited. (A) Interaction of ribbon model of α-subunit (green) of RNAP with Wol GreA consisting of NTD (cyan) and CTD (red). (B) Interaction of ribbon model β-subunit (green) of RNAP with Wol GreA having both its domains. (C) Interaction of ribbon model of β′-subunit (green) of RNAP with Wol GreA consisting of both the domains. (D) Interaction of ribbon model of σ-subunit (green) of RNAP with Wol GreA consisting of both the domains.</p

Purification and immune-localization of recombinant Wol GreA and its N and C-terminal domains.

<p>(A) Purification of Wol GreA from Rosetta strain of <i>E. coli</i> by affinity chromatography. Lane M, protein marker; Lane 1, soluble <i>E. coli</i> proteins following induction with 0.5-mM IPTG at 25°C for 6 h; Lane 2, flow through; Lane 3, washed fraction; Lanes 4-5, eluted fractions of His-tagged purified recombinant Wol GreA at 250-mM imidazole conc. (B) Purification of Wol NTD from <i>E. coli</i> expression host, Rosetta by Ni-NTA column. Lane M, protein marker; Lane 1, soluble <i>E. coli</i> proteins induced with 0.5-mM IPTG at 25°C for 6 h; Lane 2, flow through; Lane 3, washed fraction; Lanes 4–5, elution of recombinant Wol NTD at 250-mM conc. of imidazole. (C) Wol CTD purified from Rosetta strain of <i>E. coli</i>. Lane M, protein marker; Lane 1, soluble <i>E. coli</i> proteins induced under similar conditions as mentioned for Wol GreA; Lane 2, flow through; Lane 3, washed fraction; Lanes 4–5, eluted fractions of purified recombinant Wol CTD. (D) Immune-localization of recombinant Wol GreA and its domains by anti-His monoclonal antibody in Western blot. Lane M, protein marker; Lane 1, ∼18.7-kDa Wol GreA; Lane 2, ∼9-kDa Wol NTD; Lane 3, ∼10.3-kDa Wol CTD.</p

Multiple sequence alignment of deduced amino acid sequence of Wol GreA with gram-negative proteobacteria.

<p>Using ClustalW, a.a sequence of Wol GreA (wBm, reference sequence: YP_198144.1) on alignment with an array of gram-negative proteobacterial species revealed varying degrees of homology. Wol GreA revealed 90% alignment score with GreA of <i>Wolbachia</i> of <i>Onchocerca ochengi</i> (wOo, reference sequence: YP_006556205.1), 89% with <i>Wolbachia</i> of <i>Drosophila melanogaster</i> (wDm, reference sequence: NP_966418.1), 86% with <i>Wolbachia</i> of <i>Culex quinquefasciatus</i> (wCq, reference sequence: YP_001975932.1), 57% with α-proteobacteria <i>Ehrlichia canis</i> (E. can, reference sequence:YP_302733.1), 47% with <i>Rhizobium leguminosarum</i> (R. leg, reference sequence: YP_002976774.1), 46% with <i>Agrobacterium tumefaciens</i> (A. tum, reference sequence: ZP_12909003.1), 45% with rod-shaped <i>Methylobacterium radiotolerans</i> (M. rad, reference sequence: YP_001753260.1) and 44% with <i>Rickettsia africae</i> (R. afr, reference sequence: YP_002845730.1); 44% with β-proteobacteria <i>Bordetella pertussis</i> (B. per, reference sequence: NP_880910.1), and 45% with <i>Alcaligenes faecalis</i> (A. fae, reference sequence: WP_003803370.1); 41% with γ-proteobacteria <i>E. coli</i> (E. col, reference sequence: NP_417648.4), 41% with <i>Haemophilus influenza</i> (H. inf, reference sequence: NP_439483.1), 42% with <i>Yersinia pestis</i> (Y. pes, reference sequence: NP_668016.1), and 39% with <i>Vibrio cholerae</i> (V. cho, reference sequence: NP_230283.1). Identical residues are highlighted in red while the conserved amino acid changes are outlined in gray rectangular boxes. The conserved domain architecture of Wol GreA (typical of GreA superfamily) has two domains, N-terminal domain (6–78 a.a) enclosed in black box consisting of two α helices (red spiral), α1 (13–43 a.a) and α2 (52–75 a.a) separated by 3₁₀ (blue spiral) (47–49 a.a) combined together to induce nucleolytic activity of RNAP and the C terminal domain (84–163 a.a) is enclosed in green box having five β sheets (yellow arrow), β1(88–99 a.a), β2 (107–115 a.a), β3 (125–129 a.a), β4 (144–148 a.a), and β5 (153–163 a.a) verged on one side by an α-helix (131–137 a.a) shaped into a compact globular structure and executes direct binding with RNAP.</p

Determination of residual interaction between Wol GreA and α2ββ′σ subunits of Wol RNAP.

<p>(A) The protein docking study between α-subunit of RNAP and Wol GreA exhibited that CTD Lys140 donor atoms involved in hydrogen (H) bonding with acceptor atoms of Wol α subunit Thr164 and Wol CTD Asp120, Lys82 acceptor atoms form H bonding with Wol α subunit donor atoms, Asn50, Arg53, and Thr88. (B) The protein docking between β-subunit of RNAP and Wol GreA exhibited that CTD Ser105, Ser127, Ser129 donor atoms formed H bonding with Wol RNAP β subunit Asn528, Ser529, Asp 1330, Asp1331 acceptor atoms and Wol CTD Ser98, Glu116, Ser129, Val147 acceptor atoms created H bonding by interacting with Wol RNAP β subunit Lys163, Asn528, Ser529, Ser530, Asn589, and Arg1359 donor atoms. (C) Similar to α and β-subunits of RNAP, β′ also solely forms H bonding with CTD residues of Wol GreA where Ser151 donor atom formed H bond with Leu1178 acceptor atom of Wol RNAP β′ subunit and its acceptor atoms Asp104, Tyr109, Val121, Ser122, Glu153, and Phe163 involved in binding with donor atoms of Wol RNAP β′ subunit Arg129, Arg1135, Lys1195, Arg1211, Gly1297, and Arg1300. (D) The protein docking between σ-subunit of RNAP and Wol GreA exhibited that CTD acceptor atoms Glu106, Glu143 created H bonding by interacting with Lys430, Arg478 donor atoms of RNAP σ subunit.</p

Inter-molecular chemical cross-linking of Wol GreA, Wol NTD, and Wol CTD using glutaraldehyde.

<p>(A) Cross-linking study of Wol GreA. Lane M, standard protein marker; Lane 1, 5-µM Wol GreA incubated with 0.005% solution of glutaraldehyde in 50-mM phosphate buffer (pH 7.5) at 37°C for zero time interval followed by termination with 200-mM Tris-HCl (pH 8.0); Lane 2-8, 5-µM Wol GreA incubated with 0.005% glutaraldehyde for different time intervals (2 min, 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min) under identical conditions. (B) Cross-linking study of Wol NTD. Lane M, protein marker; Lane 1, 5-µM Wol NTD incubated with 0.005% glutaraldehyde in 50-mM phosphate buffer (pH 7.5) at 37°C for zero time interval and subsequently terminated with 200-mM Tris-HCl (pH 8.0); Lane 2-8, 5-µM Wol NTD incubated with 0.005% glutaraldehyde for different periods of time (2 min, 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min) under identical conditions as used for wild type. (C) Cross-linking study of Wol CTD. Lane M, protein marker; Lane 1, 5-µM Wol GreA incubated with 0.005% glutaraldehyde in phosphate buffer for zero time interval and reaction terminated with 200-mM Tris-HCl (pH 8.0); Lane 2-8, 5-µM Wol GreA incubated with 0.005% glutaraldehyde for different periods of time (2 min, 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min) under identical conditions. (D) Determination of residual interaction between Wol GreA monomers. To explore the residues of Wol GreA involved in dimerization, protein-protein docking study was performed. The monomers of GreA interact with Asp120, Val121, Ser122, Lys123, and Ser134 residues of CTD to form dimeric conformation.</p

Identification of Novel <i>S-</i>Adenosyl-l-Homocysteine Hydrolase Inhibitors through Homology-Model-Based Virtual Screening, Synthesis, and Biological Evaluation

The present study describes a successful application of computational approaches to identify novel Leishmania donovani (<i>Ld)</i> AdoHcyase inhibitors utilizing the differences for <i>Ld</i> AdoHcyase NAD⁺ binding between human and <i>Ld</i> parasite. The development and validation of the three-dimensional (3D) structures of <i>Ld</i> AdoHcyase using the L. major AdoHcyase as template has been carried out. At the same time, cloning of the <i>Ld</i> AdoHcyase gene from clinical strains, its overexpression and purification have been performed. Further, the model was used in combined docking and molecular dynamics studies to validate the binding site of NAD in <i>Ld</i>. The hierarchical structure based virtual screening followed by the synthesis of five active hits and enzyme inhibition assay has resulted in the identification of novel <i>Ld</i> AdoHcyase inhibitors. The most potent inhibitor, compound <b>5</b>, may serve as a “lead” for developing more potent <i>Ld</i> AdoHcy hydrolase inhibitors as potential antileishmanial agents