Naveen Kumar Navani

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli.

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO‐releasing compounds. A flavohaemoglobin (HMP)‐deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen‐dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35‐fold higher than the single‐domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two‐domain flavohaemoglobin. HbN‐dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5‐fold increase in NO uptake when exposed to gaseous NO, suggesting NO‐induced NOD activity in these cells. In addition, expression of plasmid‐encoded HbN of M. tuberculosis in M. smegmatis resulted in 100‐fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.

A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria

The luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens has proven to be a superb transcriptional reporter. It encodes a luciferase (LuxA and LuxB) and the enzymes that produce its substrate (LuxC, LuxD and LuxE) so cells that express the cluster emit the 490-nm light spontaneously. The sequence of these genes is AT-rich (>69%) and for this and other reasons, they are not expressed efficiently in high-GC bacteria like Streptomyces coelicolor. We therefore constructed a synthetic luxCDABE operon encoding the P. luminescens Lux proteins optimized for expression in high-GC bacteria. We tested the genes using transcriptional fusions to S. coelicolor promoters having well-established expression profiles during this organisms life cycle. The hrdB gene encodes a housekeeping sigma factor; while ramC is important for the formation of the spore-forming cells called aerial hyphae and whiE is required for the production of a grey, spore-associated pigment that is deposited in the walls of developing spores. Using these fusions we demonstrated that our synthetic lux genes are functional in S. coelicolor and that they accurately report complex developmental gene expression patterns. We suggest that this lux operon and our procedure for generating synthetic high-GC genes will be widely useful for research on high-GC bacteria.

Synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications

Silver has long been advocated as an effective antimicrobial. However, toxicity issues with silver have led to limited use of silver in nanoform, especially for food preservation. With the aim of exploring combinatorial options that could increase the antibacterial potency of silver nanoparticles and reduce the effective dosage of silver, we evaluated the extent of synergy that a combination of silver nanoparticles and an essential oil representative (cinnamaldehyde) could offer. A battery of gram-positive and gram-negative bacterial strains was utilized for antibacterial assays, and extents of synergism were calculated from fractional inhibitory concentration indices. The activity of nanoparticles was greatly enhanced when utilized in the presence of cinnamaldehyde. We observed combinatorial effects that were strongly additive against all the bacterial strains tested, and genuine synergy was found against spore forming Bacillus cereus and Clostridium perfringens – bacterial strains associated with release of cytotoxins in contaminated food and known for their persistence. Bacterial kill curve analysis revealed a very fast bactericidal action when a combination of two agents was used. The electron and atomic force microscopy also revealed extensive damage to the bacterial cell envelop in the presence of both agents. We also performed hemolysis assays to investigate and approximate the extent of toxicity exhibited by the two agents, and observed no adverse effect at the concentrations required for synergy. This study shows that safe levels of silver in nanoform in combination with essential oil component cinnamaldehyde can be effectively used for controlling the spore-forming bacterial species.

Facile biofunctionalization of silver nanoparticles for enhanced antibacterial properties, endotoxin removal, and biofilm control

Infectious diseases cause a huge burden on healthcare systems worldwide. Pathogenic bacteria establish infection by developing antibiotic resistance and modulating the host’s immune system, whereas opportunistic pathogens like Pseudomonas aeruginosa adapt to adverse conditions owing to their ability to form biofilms. In the present study, silver nanoparticles were biofunctionalized with polymyxin B, an antibacterial peptide using a facile method. The biofunctionalized nanoparticles (polymyxin B-capped silver nanoparticles, PBSNPs) were assessed for antibacterial activity against multiple drug-resistant clinical strain Vibrio fluvialis and nosocomial pathogen P. aeruginosa. The results of antibacterial assay revealed that PBSNPs had an approximately 3-fold higher effect than the citrate-capped nanoparticles (CSNPs). Morphological damage to the cell membrane was followed by scanning electron microscopy, testifying PBSNPs to be more potent in controlling the bacterial growth as compared with CSNPs. The bactericidal effect of PBSNPs was further confirmed by Live/Dead staining assays. Apart from the antibacterial activity, the biofunctionalized nanoparticles were found to resist biofilm formation. Electroplating of PBSNPs onto stainless steel surgical blades retained the antibacterial activity against P. aeruginosa. Further, the affinity of polymyxin for endotoxin was exploited for its removal using PBSNPs. It was found that the prepared nanoparticles removed 97% of the endotoxin from the solution. Such multifarious uses of metal nanoparticles are an attractive means of enhancing the potency of antimicrobial agents to control infections.

Identification of novel regulatory small RNAs in Acinetobacter baumannii.

Small RNA (sRNA) molecules are non-coding RNAs that have been implicated in regulation of various cellular processes in living systems, allowing them to adapt to changing environmental conditions. Till date, sRNAs have not been reported in Acinetobacter baumannii (A. baumannii), which has emerged as a significant multiple drug resistant nosocomial pathogen. In the present study, a combination of bioinformatic and experimental approach was used for identification of novel sRNAs. A total of 31 putative sRNAs were predicted by a combination of two algorithms, sRNAPredict and QRNA. Initially 10 sRNAs were chosen on the basis of lower E- value and three sRNAs (designated as AbsR11, 25 and 28) showed positive signal on Northern blot. These sRNAs are novel in nature as they do not have homologous sequences in other bacterial species. Expression of the three sRNAs was examined in various phases of bacterial growth. Further, the effect of various stress conditions on sRNA gene expression was determined. A detailed investigation revealed differential expression profile of AbsR25 in presence of varying amounts of ethidium bromide (EtBr), suggesting that its expression is influenced by environmental or internal signals such as stress response. A decrease in expression of AbsR25 and concomitant increase in the expression of bioinformatically predicted targets in presence of high EtBr was reverberated by the decrease in target gene expression when AbsR25 was overexpressed. This hints at the negative regulation of target genes by AbsR25. Interestingly, the putative targets include transporter genes and the degree of variation in expression of one of them (A1S_1331) suggests that AbsR25 is involved in regulation of a transporter. This study provides a perspective for future studies of sRNAs and their possible involvement in regulation of antibiotic resistance in bacteria specifically in cryptic A. baumannii.

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy. Methods/Principal Findings The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors. Conclusions/Significance The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.

Green Synthesis and Antimicrobial Potential of Silver Nanoparticles

ABSTRACT Developments in nanotechnology are leading to acceptance of this technology in day-to-day life as it continues to provide solutions and alternatives to technological, environmental, and health challenges. Central to all nanotechnology based applications are nanostructures, among which nanoparticles (NPs) are the ones most widely used. With a huge demand for various NPs, it is imperative that environmentally benign synthesis methods are developed. The majority of the existing procedures used for nanoparticle synthesis rely upon physical and chemical methods that sometimes utilize toxic and hazardous chemicals. The integration of the principles of green chemistry to nanotechnology toward the synthesis of “green” nanoparticles is a current requirement. One of the bioactive roles being investigated for NPs is their potential as antimicrobials. This review focuses on biomolecules, microbes, and phytoproducts in mediated green approaches for the synthesis of green silver nanoparticles (GSNPs) with due ...

Synthesis of glucose-mediated Ag–γ-Fe2O3 multifunctional nanocomposites in aqueous medium – a kinetic analysis of their catalytic activity for 4-nitrophenol reduction

This paper reports the synthesis of γ-Fe2O3 supported Ag nanoparticles (NPs) in aqueous medium by following a green approach. The presence of Fe2O3 in the gamma phase and silver in the nanocomposite has been confirmed by Raman spectroscopy, EDAX and XPS analyses. The presence of Ag in the nanocomposite is also indicated by UV spectroscopy. In the process of in situ generation of glucose mediated Ag NPs on the γ-Fe2O3 matrix, the size of γ-Fe2O3 nanoclusters reduced from 11.6 ± 1.6 to 9 ± 1 nm as was estimated from HRTEM analysis. Glucose served as an effective stabilizer for both Ag and γ-Fe2O3 in the nanocomposite. At lower concentrations of Ag (0.15–1.2 μM) the reduction of 4-nitrophenol (4-Nip) follows pseudo-first-order kinetics and the second order rate constant for this process was found to be 5.28 × 103 dm3 mol−1 s−1. Whereas, at higher concentrations (3.2–28.9 μM), it follows zero-order kinetics and occurred with a rate constant of 1 × 10−2 mol dm−3 s−1. The amount of silver in the nanocomposite is found to influence the kinetics of the catalytic reduction in a complex scheme following the Langmuir–Hinshelwood mechanism. The recyclability of the as-synthesized nanocomposite up to 7 cycles and the catalytic effect even at a very low silver concentration (0.15 μM) associated with high surface area and superparamagnetism suggest it to be a cost effective and environmentally friendly potential catalytic system.

Interaction of Bacteriocin-Capped Silver Nanoparticles with Food Pathogens and Their Antibacterial Effect

ABSTRACT With the emergence of multiple-drug-resistant pathogens, the antibacterial property of silver in colloidal form has emerged as a potential candidate for combating infectious diseases. A combination of antibacterial agents along with nanosilver could prove to be more potent due to broadened antibacterial spectrum with possibly lower doses. In the present study, a facile single-step green method of synthesizing silver nanoparticles functionalized with an antibacterial peptide from a food-grade lactic acid bacterium is reported. The synthesized enterocin-coated silver nanoparticles showed broad-spectrum inhibition against a battery of food-borne pathogenic bacteria without any detectable toxicity to red blood cells. The present results evince that a new category of biocide based on silver nanoparticles coated with food-grade antibacterials can be developed using simple methods.

Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis.

The thermostable, glucose tolerant β-glucosidase gene (bgl) of Glycoside hydrolase family 1, isolated from Bacillus subtilis, was cloned and overexpressed in Escherichia coli. The bgl has open reading frame of 1,407 bp, encoding 469 amino acids with predicted molecular weight of 53 kDa. The recombinant protein (BGL) was purified 10.76 fold to homogeneity with specific activity of 54.04U/mg and recovery of 38.67%. The purified BGL was optimally active at pH 6.0 and temperature 60°C. The enzyme retained more than 85% of maximum activity after 1h preincubation at 60°C. The kinetic analysis indicated that BGL has highest catalytic efficiency (Kcat/Km) against p-nitrophenyl-β-d-xylopyranoside (654.58 mM(-1)s(-1)) followed by p-nitrophenyl-β-d-glucopyranoside (292.53 mM(-1)s(-1)) and p-nitrophenyl-β-d-galactopyranoside (61.17 mM(-1)s(-1)). The Ki value for glucose and δ-gluconolactone was determined to be 1.9 mM and 0.018 mM, respectively. The BGL exhibited high tolerance against detergents and organic solvents. The homology modeling revealed that protein has 19 α-helices and 4 β-sheets and adopted (α/β)8 TIM barrel structure. Substrate docking and LigPlot analysis depicted the amino acids of active site involved in hydrogen bonding and hydrophobic interactions with substrates. The efficient BGL secretion with exploration of structural and functional relationship offer vistas for large scale production and various industrial applications.