Ranjana Pathania

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.

Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.

ABSTRACT Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 μM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.

Chemical genomics in Escherichia coli identifies an inhibitor of bacterial lipoprotein targeting

One of the most significant hurdles to developing new chemical probes of biological systems and new drugs to treat disease is that of understanding the mechanism of action of small molecules discovered with cell-based small-molecule screening. Here we have assembled an ordered, high-expression clone set of all of the essential genes from Escherichia coli and used it to systematically screen for suppressors of growth inhibitory compounds. Using this chemical genomic approach, we demonstrate that the targets of well-known antibiotics can be identified as high copy suppressors of chemical lethality. This approach led to the discovery of MAC13243, a molecule that belongs to a new chemical class and that has a unique mechanism and promising activity against multidrug-resistant Pseudomonas aeruginosa. We show that MAC13243 inhibits the function of the LolA protein and represents a new chemical probe of lipoprotein targeting in bacteria with promise as an antibacterial lead with Gram-negative selectivity.

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.

Diversity of bacterial isolates during full scale rotary drum composting.

Bacterial diversity of full scale rotary drum composter from biodegradable organic waste samples were analyzed through two different approaches, i.e., Culture dependent and independent techniques. Culture-dependent enumerations for indigenous population of bacterial isolates mainly total heterotrophic bacteria (Bacillus species, Pseudomonas species and Enterobacter species), Fecal Coliforms, Fecal Streptococci, Escherichia coli, Salmonella species and Shigella species showed reduction during the composting period. On the other hand, Culture-independent method using PCR amplification of specific 16S rRNA sequences identified the presence of Acinetobacter species, Actinobacteria species, Bacillus species, Clostridium species, Hydrogenophaga species, Butyrivibrio species, Pedobacter species, Empedobactor species and Flavobacterium species by sequences clustering in the phylogenetic tree. Furthermore, correlating physico-chemical analysis of samples with bacterial diversity revealed the bacterial communities have undergone changes, possibly linked to the variations in temperature and availability of new metabolic substrates while decomposing organics at different stages of composting.

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.

Microbial diversity during Rotary Drum and Windrow Pile composting.

This study investigates the prevailing microbial communities during the composting of vegetable waste, cattle manure and saw dust, in a household (250 l) batch scale Rotary Drum composter and Windrow Pile. Physico‐chemical parameters were analyzed to study the organic matter transformations. Total organic matter reduced from 63.8% to 36.2% in rotary drum and 39.6% in windrow pile composting. The C/N ratio decreased from 26.52 to 8.89 and 14.33 in rotary drum and windrow pile composting. The indigenous population of total heterotrophic bacteria decreased in rotary drum and windrow pile composting after 20 days. However, total fungal load initially increased within initial 4 days, then subsequently reduced in final composts. The average number of fecal coliforms and fecal Streptococci showed decrement with time, in both composting systems. Escherichia coli and Salmonella species number deduced during the study. Composting cycle started with Gram positive rods but ended up with the dominance of Gram negative bacilli shaped bacteria. Transformation of organic compounds during the biodegradation of organic waste, difference in the utilization of nutrients (organic matter) by the different group of microbes and high temperature could be cited as a possible reason of the above changes. Scanning electron microscopy has been used to obtain the surface structures of the cultured mycoflora. Results of the study revealed that higher diversity of microbes prevailed in rotary drum as compared to windrow pile, yielding more stable and pathogenic free compost in lesser period of composting. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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 ...