Publications by authors named "Timsy Bhando"

6 Publications

  • Page 1 of 1

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Biotechnol Appl Biochem 2021 Apr 20;68(2):213-220. Epub 2020 May 20.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, k /K , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.
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http://dx.doi.org/10.1002/bab.1912DOI Listing
April 2021

Antibacterial properties and in vivo efficacy of a novel nitrofuran, IITR06144, against MDR pathogens.

J Antimicrob Chemother 2020 02;75(2):418-428

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India.

Objectives: The emergence of MDR Gram-negative pathogens and increasing prevalence of chronic infections presents an unmet need for the discovery of novel antibacterial agents. The aim of this study was to evaluate the biological properties of a small molecule, IITR06144, identified in a phenotypic screen against the Gram-negative model organism Escherichia coli.

Methods: A small-molecule library of 10956 compounds was screened for growth inhibition against E. coli ATCC 25922 at concentration 50 μM. MICs of lead compounds were determined by the broth microdilution method. Time-kill kinetics, anti-persister activity, spontaneous frequency of resistance, biofilm inhibition and disruption were assessed by standard protocols. Resistant mutants were generated by serial passaging followed by WGS. In vitro toxicity studies were carried out via the MTT assay. In vivo toxicity and efficacy in a mouse model were also evaluated.

Results: IITR06144 was identified as the most promising candidate amongst 29 other potential antibacterial leads, exhibiting the lowest MIC, 0.5 mg/L. IITR06144 belongs to the nitrofuran class and exhibited broad-spectrum bactericidal activity against most MDR bacteria, including the 'priority pathogen', carbapenem-resistant Acinetobacter baumannii. IITR06144 retained its potency against nitrofurantoin-resistant clinical isolates. It displayed anti-persister, anti-biofilm activity and lack of spontaneous resistance development. IITR06144 demonstrated a large therapeutic index with no associated in vitro and in vivo toxicity.

Conclusions: In the light of excellent in vitro properties displayed by IITR06144 coupled with its considerable in vivo efficacy, further evaluation of IITR06144 as a therapeutic lead against antibiotic-resistant infections is warranted.
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http://dx.doi.org/10.1093/jac/dkz428DOI Listing
February 2020

The unusual glycine-rich C terminus of the RNA chaperone Hfq plays an important role in bacterial physiology.

J Biol Chem 2018 08 12;293(35):13377-13388. Epub 2018 Jul 12.

From the Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India and

is a Gram-negative nosocomial pathogen that causes soft tissue infections in patients who spend a long time in intensive care units. This recalcitrant bacterium is very well known for developing rapid drug resistance, which is a combined outcome of its natural competence and mobile genetic elements. Successful efforts to treat these infections would be aided by additional information on the physiology of Toward that end, we recently reported on a small RNA (sRNA), AbsR25, in this bacterium that regulates the genes of several efflux pumps. Because sRNAs often require the RNA chaperone Hfq for assistance in binding to their cognate mRNA targets, we identified and characterized this protein in The homolog in is a large protein with an extended C terminus unlike Hfqs in other Gram-negative pathogens. The extension has a compositional bias toward glycine and, to a lower extent, phenylalanine and glutamine, suggestive of an intrinsically disordered region. We studied the importance of this glycine-rich tail using truncated versions of Hfq in biophysical assays and complementation of an deletion mutant, finding that the tail was necessary for high-affinity RNA binding. Further tests implicate Hfq in important cellular processes of like metabolism, drug resistance, stress tolerance, and virulence. Our findings underline the importance of the glycine-rich C terminus in RNA binding, ribo-regulation, and auto-regulation of Hfq, demonstrating this hitherto overlooked protein motif to be an indispensable part of the Hfq.
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http://dx.doi.org/10.1074/jbc.RA118.002921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120210PMC
August 2018

Fosfomycin resistance in Acinetobacter baumannii is mediated by efflux through a major facilitator superfamily (MFS) transporter-AbaF.

J Antimicrob Chemother 2017 01 20;72(1):68-74. Epub 2016 Sep 20.

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India

Objectives: To decipher the function of A1S_1331, named AbaF (Acinetobacter baumannii Fosfomycin efflux), one of the primary targets of AbsR25, a small RNA of A. baumannii.

Methods: abaF was cloned in a multicopy plasmid and expressed from its native promoter in an efflux-deficient strain-Escherichia coli KAM32. Drug susceptibility, accumulation and efflux of ethidium bromide (EtBr) were determined in this strain. abaF was disrupted in A. baumannii using homologous recombination and its effect on drug susceptibility, biofilm formation and virulence was studied. Expression of abaF was followed by quantitative PCR in fosfomycin-challenged A. baumannii and fosfomycin-resistant mutants of A. baumannii. Expression of abaF in clinical strains of A. baumannii was determined by RT-PCR.

Results: Expression of abaF in E. coli KAM32 resulted in increased resistance to fosfomycin. Lower accumulation and higher efflux of EtBr from this strain confirmed the role of AbaF as an efflux pump. Disruption of abaF in A. baumannii caused an increase in fosfomycin susceptibility and a decrease in biofilm formation and virulence. The expression of abaF was higher in A. baumannii cells exposed to fosfomycin and in cells resistant to higher concentrations of fosfomycin. The clinically relevant strains of A. baumannii also tested positive for the expression of abaF.

Conclusions: The results of this study suggest that efflux is an important mechanism of fosfomycin resistance and AbaF is involved in fosfomycin resistance in A. baumannii. AbaF also seems to play a role in biofilm formation and virulence of A. baumannii.
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http://dx.doi.org/10.1093/jac/dkw382DOI Listing
January 2017

Bilobed shape of PadA reveals the connectivity from single to multi-domain bacterial plasminogen activators.

Int J Biol Macromol 2015 18;78:370-8. Epub 2015 Apr 18.

CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India. Electronic address:

The bacterial plasminogen activator, PadA activates bovine, ovine and caprine plasminogen but remains inert toward human plasminogen. It shows high sequence homology with human plasminogen activator, staphylokinase (SAK) but generates active-site in bovine plasminogen non-proteolytically, similar to streptokinase (SK). To examine the structural requirements for the function of this unique cofactor, attempts were made to visualize solution structure of the PadA using small-angle X-ray scattering (SAXS) data and compare its shape profile with structural models based on crystal structures of staphylokinase and streptokinase domains. The bilobal shape solved for the PadA matched closely with the structural model of α-domain of SK rather than its sequence homolog, SAK. The SAXS based solution structure of the PadA exhibited an extra volume and high mobility around Y(90)DKAEK(95) and P(104)ITES(108) loop regions that were found to play a crucial role in its cofactor function. Structure and sequence analysis of bacterial cofactors and mammalian plasminogens displayed evolutionary conservation of crucial complimentary amino acids required for making a functional binary activator complex between bacterial plasminogen activators and their cognate partner plasminogen. These studies highlighted the importance of structure-function related evolutionary strategies adopted by bacteria for exploiting mammalian plasminogen activation system and its understanding may help in designing and the development of new thrombolytic agents for clinical interventions.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.04.019DOI Listing
February 2016

Fibrin-targeted plasminogen activation by plasminogen activator, PadA, from Streptococcus dysgalactiae.

Protein Sci 2014 Jun 5;23(6):714-22. Epub 2014 Apr 5.

CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh, 160036, India.

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three-domain streptokinase (SK) and two-domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2-antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single-domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single-domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single-domain plasminogen activator that activates bovine plasminogen in a fibrin-targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin-specific plasminogen activators for thrombolytic therapy.
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http://dx.doi.org/10.1002/pro.2455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093948PMC
June 2014