Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

Amine-functionalized multiwall carbon nanotubes impart osteoinductive and bactericidal properties in poly(epsilon-caprolactone) composites

Poly(epsilon-caprolactone) (PCL) is an aliphatic polyester widely used for biomedical applications but lacks the mechanical properties desired for many load-bearing orthopedic applications. The objective of this study was to prepare and characterize PCL composites incorporating multiwall carbon nanotubes (MWNTs) with different surface functional groups. PCL composites were prepared by melt-mixing with three different types of MWNTs: pristine (pMWNT), amine functionalized (aMWNT), and carboxyl functionalized (cMWNT). Melt rheology and scanning electron microscopy indicated good dispersion of the nanotubes in the matrix. Tensile strength and elastic modulus of the polymer was significantly increased by the incorporation of MWNTs and further enhanced by favorable interactions between PCL and aMWNTs. Thermal analysis revealed that MWNTs act as heterogeneous nucleation sites for crystallization of PCL and increase polymer crystallinity. Incorporation of functionalized MWNTs increased the surface water wettability of PCL. Osteoblast proliferation and differentiation was significantly enhanced on functionalized composites. aMWNT composites also exhibited the best bactericidal response. This study demonstrates that surface functionalization of MWNTs profoundly influences the properties of PCL and amine-functionalization offers the optimal combination of mechanical properties, osteogenesis and antimicrobial response. These results have important implications for designing nanocomposites for use in orthopedics.

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.

Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.

Bioactive compounds comprising secondary metabolites produced by endophytic fungi have wide applications in pharmacology and agriculture. Isolation, characterisation and evaluation of biological activities of secondary metabolites were carried out from Cochliobolus kusanoi an endophytic fungus of Nerium oleander L. The fungus was identified based on 18S rDNA sequence analysis. There are no reports available on the compounds of C. kusanoi hence, antimicrobial metabolite produced by this fungus was extracted and purified by fractionation using hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Out of all the solvent fractions, the methanol fraction exhibited better antimicrobial activity which was further purified and characterised as oosporein. Oosporein from C. kusanoi exhibited broad spectrum in vitro antimicrobial, antioxidant and cytotoxic activities. The characterisation and antioxidant activity of oosporein from C. kusanoi are reported for the first time.

Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence

Background: Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. Results: We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. Conclusions: A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would provide insight into conditions which are encountered by this intracellular enteric pathogen during the course of infection. This would further contribute in identifying novel targets for antimicrobial agents. (C) 2014 Elsevier Ltd. All rights reserved.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and Mycobacterium tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the Mycobacterium tuberculosis and Mycobacterium smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 x 10(-6)m Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.

Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study

The de novo purine biosynthesis is one of the highly conserved pathways among all organisms and is essential for the cell viability. A clear understanding of the enzymes in this pathway would pave way for the development of antimicrobial and anticancer drugs. Phosphoribosylaminoimidazole-succinocar boxamide (SAICAR) synthetase is one of the enzymes in this pathway that catalyzes ATP dependent ligation of carboxyaminoimidazole ribotide (CAIR) with L-aspartate (ASP). Here, we describe eight crystal structures of this enzyme, in C222(1) and H3 space groups, bound to various substrates and substrate mimics from a hyperthermophilic archaea Pyrococcus horikoshii along with molecular dynamics simulations of the structures with substrates. Complexes exhibit minimal deviation from its apo structure. The CAIR binding site displays a preference for pyrimidine nucleotides. In the ADP.TMP-ASP complex, the ASP binds at a position equivalent to that found in Saccharomyces cerevisiae structure (PDB: 2CNU) and thus, clears the ambiguity regarding ASP's position. A possible mode for the inhibition of the enzyme by CTP and UTP, observed earlier in the yeast enzyme, is clearly illustrated in the structures bound to CMP and UMP. The ADP.Mg2+.PO4.CD/MP complex having a phosphate ion between the ATP and CAIR sites strengthens one of the two probable pathways (proposed in Escherichia coli study) of catalytic mechanism and suggests the possibility of a phosphorylation taking place before the ASP's attack on CAIR. Molecular dynamic simulations of this enzyme along with its substrates at 90 degrees C reveal the relative strengths of substrate binding, possible antagonism and the role of Mg2+ ions. (C) 2015 Elsevier Inc. All rights reserved.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

An ultrastable conjugate of silver nanoparticles and protein formed through weak interactions

In recent years, silver nanoparticles (AgNPs) have attracted significant attention owing to their unique physicochemical, optical, conductive and antimicrobial properties. One of the properties of AgNPs which is crucial for all applications is their stability. In the present study we unravel a mechanism through which silver nanoparticles are rendered ultrastable in an aqueous solution in complex with the protein ubiquitin (Ubq). This involves a dynamic and reversible association and dissociation of ubiquitin from the surface of AgNP. The exchange occurs at a rate much greater than 25 s(-1) implying a residence time of <40 ms for the protein. The AgNP-Ubq complex remains stable for months due to steric stabilization over a wide pH range compared to unconjugated AgNPs. NMR studies reveal that the protein molecules bind reversibly to AgNP with an approximate dissociation constant of 55 mu M and undergo fast exchange. At pH > 4 the positively charged surface of the protein comes in contact with the citrate capped AgNP surface. Further, NMR relaxation-based experiments suggest that in addition to the dynamic exchange, a conformational rearrangement of the protein takes place upon binding to AgNP. The ultrastability of the AgNP-Ubq complex was found to be useful for its anti-microbial activity, which allowed the recycling of this complex multiple times without the loss of stability. Altogether, the study provides new insights into the mechanism of protein-silver nanoparticle interactions and opens up new avenues for its application in a wide range of systems.

Novel AgBr/Ag3PO4 Decorated Ceria Nanoflake Composites for Enhanced Photocatalytic Activity toward Dyes and Bacteria under Visible Light

The aim of this study was to develop heterogeneous visible light active photocatalysts using AgBr and Ag3PO4 using CeO2 nanoflakes as an efficient substrate. Ascorbic acid was employed as a fuel to synthesize fine ceria nanoflakes by a facile solution combustion process. AgBr and Ag3PO4 were decorated on ceria to prepare AgBr/Ag3PO4/ceria nanocomposites. The structure of the composite was determined by X-ray diffraction analysis. Novel flakelike morphology was revealed using electron microscopy techniques. The nanocomposites exhibit excellent photocatalytic activity under visible light compared to pristine ceria nanoparticles. The nanocomposite catalyst particles degraded both anionic and cationic dyes. It also exhibited efficient antimicrobial activity under visible light. The AgBr/Ag3PO4/ceria nanocomposite was characterized using X-ray diffraction analysis, diffuse reflectance spectroscopy, electron microscopy, BET surface area analysis, and X-ray photoelectron spectroscopy, and the reasons for enhanced photocatalytic activity were elucidated. The presence of silver based semiconductors on ceria has shown to decrease charge recombination through photoluminescence analysis that attributed for enhanced photocatalytic activity. The AgBr/Ag3PO4/ceria nanocomposite has shown a stable performance after many repeated cycles.

Synthesis of Novel Benzofuran-Gathered C-2,4,6-substituted Pyrimidine Derivatives Conjugated by Sulfonyl Chlorides: Orally Bioavailable, Selective, Effective Antioxidants and Antimicrobials Drug Candidates

In the present study, we have made an effort to develop the novel synthetic antioxidants and antimicrobials with improved potency. The novel benzofuran-gathered C-2,4,6-substituted pyrimidine derivatives 5a, 5b, 5c, 5d, 5e, 5f, 6a, 6b, 6c, 6d, 6e, 6f, 7a, 7b, 7c, 7d, 7e, 7f, 8a, 8b, 8c, 8d, 8e, 8f, 9a, 9b, 9c, 9d, 9e, 9f were synthesized by simple and efficient four-step reaction pathway. Initially, o-alkyl derivative of salicylaldehyde readily furnish corresponding 2-acetyl benzofuran 2 in good yield, upon the treatment with potassium tertiary butoxide in the presence of molecular sieves. Further, Claisen-Schmidt condensation with aromatic aldehydes via treatment with thiourea followed by coupling reaction with different sulfonyl chlorides afforded target compounds. The structures of newly synthesized compounds were confirmed by IR, H-1 NMR, C-13 NMR, mass, and elemental analysis and further screened for their antioxidant and antimicrobial activities. The results showed that the synthesized compounds 8b, 8e, 9b, and 9e produced significant antioxidant activity with 50% inhibitory concentration higher than that of reference, whereas compounds 7d and 7c produced dominant antimicrobial activity at concentrations 1.0 and 0.5mg/mL compared with standard Gentamicin and Nystatin, respectively.

Differential viability response of prokaryotes and eukaryotes to high strength pulsed magnetic stimuli

The present study examines the efficacy of a high strength pulsed magnetic field (PMF) towards bacterial inactivation in vitro, without compromising eukaryotic cell viability. The differential response of prokaryotes Staphylococcus aureus (MESA), Staphylococcus epidermidis, and Escherichia coli], and eukaryotes C2C12 mouse myoblasts and human mesenchymal stem cells, hMSCs] upon exposure to varying PMF stimuli (1-4 T, 30 pulses, 40 ms pulse duration) is investigated. Among the prokaryotes, similar to 60% and similar to 70% reduction was recorded in the survival of staphylococcal species and E. coli, respectively at 4 T PMF as evaluated by colony forming unit (CPU) analysis and flow cytometry. A 2-5 fold increase in intracellular ROS (reactive oxygen species) levels suggests oxidative stress as the key mediator in PMF induced bacterial death/injury. The 4 T PMF treated staphylococci also exhibited longer doubling times. Both TEM and fluorescence microscopy revealed compromised membranes of PMF exposed bacteria. Under similar PMF exposure conditions, no immediate cytotoxicity was recorded in C2C12 mouse myoblasts and hMSCs, which can be attributed to the robust resistance towards oxidative stress. The ion interference of iron containing bacterial proteins is invoked to analytically explain the PMF induced ROS accumulation in prokaryotes. Overall, this study establishes the potential of PMF as a bactericidal method without affecting eukaryotic viability. This non-invasive stimulation protocol coupled with antimicrobial agents can be integrated as a potential methodology for the localized treatment of prosthetic infections. (C) 2015 Elsevier B.V. All rights reserved.

Stimuli-responsive weak polyelectrolyte multilayer films: A thin film platform for self triggered multi-drug delivery

Polyelectrolyte multilayer (PEM) thin film composed of weak polyelectrolytes was designed by layer-by-layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) for multi-drug delivery applications. Environmental stimuli such as pH and ionic strength showed significant influence in changing the film morphology from pore-free smooth structure to porous structure and favored triggered release of loaded molecules. The film was successfully loaded with bovine serum albumin (BSA) and ciprofloxacin hydrochloride (CH) by modulating the porous polymeric network of the film. Release studies showed that the amount of release could be easily controlled by changing the environmental conditions such as pH and ionic strength. Sustained release of loaded molecules was observed up to 8 h. The fabricated films were found to be biocompatible with epithelial cells during in-vitro cell culture studies. PEM film reported here not only has the potential to be used as self-responding thin film platform for transdermal drug delivery, but also has the potential for further development in antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2015 Elsevier B.V. All rights reserved.