Browsing by Author "Lav Kumar Jaiswal"

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A comparative analysis of mycobacterial ribonucleases: Towards a therapeutic novel drug target
(Elsevier B.V., 2024) Lav Kumar Jaiswal; Rakesh Kumar Singh; Tanmayee Nayak; Anuja Kakkar; Garima Kandwal; Vijay Shankar Singh; Ankush Gupta
Bacterial responses to continuously changing environments are addressed through modulation of gene expression at the level of transcription initiation, RNA processing and/or decay. Ribonucleases (RNases) are hydrolytic or phosphorolytic enzymes involved in a majority of RNA metabolism reactions. RNases play a crucial role in RNA degradation, either independently or in collaboration with various trans-acting regulatory factors. The genus Mycobacterium consists of five subgenera: Mycobacteroides, Mycolicibacterium, Mycobacterium, Mycolicibacter and Mycolicibacillus, which include 63 fully sequenced species (pathogenic/non-pathogenic) to date. These include 13 different RNases, among which 5 are exonucleases (RNase PH, PNPase, RNase D, nano-RNases and RNase AS) and 8 are endonucleases (RNase J, RNase H, RNase P, RNase III, RNase BN, RNase Z, RNase G and RNase E), although RNase J and RNase BN were later identified to have exoribonuclease functions also. Here, we provide a detailed comparative insight into the Escherichia coli and mycobacterial RNases with respect to their types, phylogeny, structure, function, regulation and mechanism of action, with the main emphasis on RNase E. Among these 13 different mycobacterial RNases, 10 are essential for cell survival and have diverse structures hence, they are promising drug targets. RNase E is also an essential endonuclease that is abundant in many bacteria, forms an RNA degradosome complex that controls central RNA processing/degradation and has a conserved 5′ sensor domain/DNase-I like region in its RNase domain. The essential mycobacterial RNases especially RNase E provide a potential repertoire of drug targets that can be exploited for inhibitor/modulator screening against many deadly mycobacterial diseases. © 2024 The Authors
Biopesticides: Current status and future prospects in India
(Springer Singapore, 2019) Yashoda Nandan Tripathi; Kumari Divyanshu; Sunil Kumar; Lav Kumar Jaiswal; Atif Khan; Hareram Birla; Ankush Gupta; Surya Pratap Singh; Ram Sanmukh Upadhyay
For over a century, chemical control of pests is a common practice in agriculture. The average reduction in global crop loss due to use of pesticides is around ~39%. The postharvest losses and quality decline caused by storage pests are major problems in a subtropical country like India. So, the farmers have relied heavily on the use of chemical pesticides to improve their crop production, which is now paying a huge toll on the human health and environment. Though the chemical pesticides are very effective, what concerns over their use is their effect on soil and environment and presence of residue in food products. Another major issue is the development of resistance in the pests. Therefore, the use of biopesticides to control pests is now preferred over synthetic pesticides because of their pest control ability and diverse mode of actions which helps in avoiding resistance development in the pests. In a country like India with a huge diversity of plants, there is an urgent need for identifying new biopesticides which can serve the purpose of pest control. India needs to develop its own biocontrol agents (BCA) because it will be cost-effective and also environment-friendly. Major hurdle in the development and use of new biopesticides in India is the commercialization process. The farmers are reluctant to use the new products because of high cost and no practical knowledge. © Springer Nature Singapore Pte Ltd. 2020.
Characterization of a novel virulent mycobacteriophage Kashi-SSH1 (KSSH1) depicting genus-specific broad-spectrum anti-mycobacterial activity
(Elsevier Inc., 2025) Tanmayee Nayak; Anuja Kakkar; Lav Kumar Jaiswal; Garima Kandwal; Anand K. Singh; Louise M. Temple; Ankush Gupta
Aim: Tuberculosis (TB) is one of the leading infectious disease causing mortality in the world and the rise of drug resistance; multi-drug resistance (MDR) and extensive-drug resistance (XDR) has added to extra complicacy of the disease. In this scenario, phage therapy has emerged as a potential treatment option against drug-sensitive/-resistant strains. Materials and methods: The mycobacteriophage Kashi-SSH1 (KSSH1) was isolated from soil sample and was genomically, phenotypically, and functionally characterized. It includes genome assembly/annotation, transmission electron microscopy, multiplicity of infection (MOI), one-step growth curve, temperature/pH stability, confocal microscopy, host range determination and host growth reduction assays. Key findings: KSSH1 is a novel polyvalent virulent mycobacteriophage from the Myoviridae family, classified under cluster C1 with a 155,659 bp genome carrying key lysis genes—Holliday junction resolvase, Holin, Lysin A, and Lysin B, has an optimal MOI of 0.01, a 60-min latent period, and a burst size of 200 phages/bacterial cell. It remains stable up to 55 °C and within pH 7–10, exhibiting broad-spectrum activity against Mycobacterium species, like M. fortuitum (opportunistic pathogen), M. tuberculosis H37Ra (attenuated pathogen), and M. smegmatis, but not non-mycobacterial hosts. KSSH1 exhibits comparable growth inhibition of M. smegmatis like the antibiotics isoniazid and rifampicin as compared to the control, in liquid cultures for over 50 h without regrowth. Significance: KSSH1 exhibits strong lytic activity against various Mycobacterium species, lacks lysogeny-associated genes like integrases/transcriptional repressors, antibiotic resistance and virulence genes and remains stable from 4 °C to 37 °C and pH 8–10 ensuring safety/stability making it an ideal candidate for therapeutic use. © 2025
Comprehensive kinetic characterization and inhibitor study of Ribonuclease E, a key enzyme of RNA metabolism from Mycobacterium smegmatis
(Elsevier B.V., 2025) Lav Kumar Jaiswal; Tanmayee Nayak; Anuja Kakkar; Garima Kandwal; Shubham Vashishtha; Kritika Singh; Bishwajit Kundu; Ankush Gupta
Ribonuclease E (RNase E) in Mycobacterium species is an essential gene that plays a crucial role in RNA metabolism. This is the first detailed kinetic characterization of recombinant RNase E and its deletion mutants, along with virtual high-throughput ligand screening (vHTS) and in vitro inhibitor studies from any Mycobacterium species. Recombinant wild-type RNase E and its deletion mutants of Mycobacterium smegmatis (M. smegmatis) were cloned, expressed and purified using Ni+2-NTA affinity chromatography. The optimal expression of recombinant His-RNase E was fine-tuned by adjusting various parameters, that included choice of Codon Plus(DE3) as expression host strain at 33 °C temperature and 0.1 mM IPTG concentration. Semi-quantitative RNase E activity was analyzed by an agarose gel-based assay using M. smegmatis total RNA as native substrate, depicting time-dependent degradation by the purified recombinant proteins. Kinetic characterization of wild-type and RNase E deletion mutants was performed by a liquid assay using methylene blue-RNA intercalation by monitoring the change in absorbance at 690 nm. The optimized enzyme concentration of 0.4 μg ml−1 was utilized throughout the assay and the wild-type enzyme depicted maximum activity at 50 °C temperature and pH 7. The wild-type RNase E activity increased by 40 % and 8 % in presence of 5 mM Mn+2 and Mg+2 cations respectively. Maximum velocity (Vmax) of the wild-type was 11.89 ± 0.1057 μg ml−1 min−1 and Km was 28.26 ± 1.828 μg ml−1, comparable with the N-terminal deletion mutant P-3 while the C-terminal deletion mutant P-2 demonstrates ∼3 folds lower Vmax and ∼ 3 folds higher Km as compared to the wild-type RNase E P-1. Being an essential gene, principle regulator of RNA metabolism and a promising antibacterial drug target, in silico vHTS of its highly conserved RNase domain and Molecular Dynamics (MD) simulations identified Sirolimus and levothyroxine as two top hit inhibitors. In vitro RNA degradation assay further confirmed Sirolimus as a non-competitive inhibitor of RNase E with Ki of 1.0 μM. In future, Sirolimus can be developed as potent anti-mycobacterial drug targetting RNase E after further modifications/ derivatization studies. © 2025
Effects of Salt Stress on Nutrient Cycle and Uptake of Crop Plants
(wiley, 2021) Lav Kumar Jaiswal; Prabhakar Singh; Rakesh Kumar Singh; Tanamyee Nayak; Yashoda Nandan Tripathi; Ram Sanmukh Upadhyay; Ankush Gupta
The biogeochemical cycling of elements involves the interactions between the biosphere and the surroundings in the form of cycling of elements throughout the different spheres viz., atmosphere, hydrosphere, and lithosphere. This chapter elucidates the effects of salinity stress on biogeochemical cycle and soil microorganisms that directly or indirectly affect the crop production. Nutrient uptake, primary productivity, and other biological processes are widely affected by nutrient limitation in terrestrial ecosystems. Nutrient cycling is one of the most important phenomena that occur in an ecosystem. The nutrient cycle represents the use, movement, and recycling of nutrients in the environment. Salt stress has an overall negative effect on carbon cycle which is implemented by its effect on various components of carbon cycle viz. photosynthesis, and microbial decomposers. Oxygen and water cycle or hydrological cycle are two biogeochemical cycles through which oxygen and water moves through biotic and abiotic components of earth and are essential for survival of life. © 2022 John Wiley & Sons Ltd. All rights reserved.
Engineered bacteriophages: A panacea against pathogenic and drug resistant bacteria
(Elsevier Ltd, 2024) Anuja Kakkar; Garima Kandwal; Tanmayee Nayak; Lav Kumar Jaiswal; Amit Srivastava; Ankush Gupta
Antimicrobial resistance (AMR) is a major global concern; antibiotics and other regular treatment methods have failed to overcome the increasing number of infectious diseases. Bacteriophages (phages) are viruses that specifically target/kill bacterial hosts without affecting other human microbiome. Phage therapy provides optimism in the current global healthcare scenario with a long history of its applications in humans that has now reached various clinical trials. Phages in clinical trials have specific requirements of being exclusively lytic, free from toxic genes with an enhanced host range that adds an advantage to this requisite. This review explains in detail the various phage engineering methods and their potential applications in therapy. To make phages more efficient, engineering has been attempted using techniques like conventional homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, CRISPY-BRED/Bacteriophage Recombineering with Infectious Particles (BRIP), chemically accelerated viral evolution (CAVE), and phage genome rebooting. Phages are administered in cocktail form in combination with antibiotics, vaccines, and purified proteins, such as endolysins. Thus, phage therapy is proving to be a better alternative for treating life-threatening infections, with more specificity and fewer detrimental consequences. © 2024 The Authors
Expression, Purification, and in Silico Characterization of Mycobacterium smegmatis Alternative Sigma Factor SigB
(Hindawi Limited, 2022) Rakesh Kumar Singh; Lav Kumar Jaiswal; Tanmayee Nayak; Ravindra Singh Rawat; Sanjit Kumar; Sachchida Nand Rai; Ankush Gupta
Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA (σ70) but dispensable for growth in both Mycobacterium smegmatis (Msmeg) and Mycobacterium tuberculosis (Mtb). It is involved in general stress responses including heat, oxidative, surface, starvation stress, and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive extra cytoplasmic function (ECF) sigma factors (SigH, SigE, SigL) and SigF involved in multiple signaling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of E. coli, viz., (BL21 (DE3), C41 (DE3), and CodonPlus (DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time duration. We achieved the maximum expression of SigB at 25°C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA that is carried out using homology modeling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Msmeg and SigB. This effort provides a platform for pulldown assay, structural, and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signaling networks along with its regulation. © 2022 Rakesh Kumar Singh et al.
Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis
(Springer Science and Business Media Deutschland GmbH, 2023) Lav Kumar Jaiswal; Rakesh Kumar Singh; Tanmayee Nayak; Vinay Kumar Singh; Ankush Gupta
Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies. © 2023, The Author(s), under exclusive licence to Shiraz University.
Isolation and characterization of a novel mycobacteriophage Kashi-VT1 infecting Mycobacterium species
(Frontiers Media SA, 2023) Tanmayee Nayak; Anuja Kakkar; Rakesh Kumar Singh; Lav Kumar Jaiswal; Anand Kumar Singh; Louise Temple; Ankush Gupta
Mycobacteriophages are viruses that infect members of genus Mycobacterium. Because of the rise in antibiotic resistance in mycobacterial diseases such as tuberculosis, mycobacteriophages have received renewed attention as alternative therapeutic agents. Mycobacteriophages are highly diverse, and, on the basis of their genome sequences, they are grouped into 30 clusters and 10 singletons. In this article, we have described the isolation and characterization of a novel mycobacteriophage Kashi-VT1 (KVT1) infecting Mycobacterium >smegmatis mc2 155 (M. smegmatis) and Mycobacterium fortuitum isolated from Varanasi, India. KVT1 is a cluster K1 temperate phage that belongs to Siphoviridae family as visualized in transmission electron microscopy. The phage genome is 61,010 base pairs with 66.5% Guanine/Cytosine (GC) content, encoding 101 putative open reading frames. The KVT1 genome encodes an immunity repressor, a tyrosine integrase, and an excise protein, which are the characteristics of temperate phages. It also contains genes encoding holin, lysin A, and lysin B involved in host cell lysis. The one-step growth curve demonstrated that KVT1 has a latency time of 90 min and an average burst size of 101 phage particles per infected cell. It can withstand a temperature of up to 45°C and has a maximum viability between pH 8 and 9. Some mycobacteriophages from cluster K are known to infect the pathogenic Mycobacterium tuberculosis (M. tuberculosis); hence, KVT1 holds potential for the phage therapy against tuberculosis, and it can also be engineered to convert into an exclusively lytic phage. Copyright © 2023 Nayak, Kakkar, Singh, Jaiswal, Singh, Temple and Gupta.