@article {8097, title = {Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Res Microbiol}, year = {2023}, month = {2023 Feb 02}, pages = {104039}, abstract = {

OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, β-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both, hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion-pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.

}, issn = {1769-7123}, doi = {10.1016/j.resmic.2023.104039}, author = {Bhowmik, Purnendu and Bharatham, Nagakumar and Murakami, Satoshi and Ramachandran, Vasanthi and Datta, Santanu} } @article {2598, title = {Validated In Silico Model for Biofilm Formation in Escherichia coli [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {ACS Synthetic Biology}, year = {2022}, month = {01/2022}, abstract = {

Using\ Escherichia coli\ as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on \~{}300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic\ E. coli\ were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.

}, doi = {10.1021/acssynbio.1c00445}, url = {https://doi.org/10.1021/acssynbio.1c00445}, author = {Bhowmik, Purnendu and Rajagopal, Sreenath and Hmar, Rothangamawi Victoria and Singh, Purnima and Saxena, Pragya and Amar, Prakruthi and Thomas, Teby and Ravishankar, Rajani and Nagaraj, Savitha and Katagihallimath, Nainesh and Sarangapani, Ramanujan Kadambi and Ramachandran, Vasanthi and Datta, Santanu} } @article {1861, title = {Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae [Bugworks, a C-CAMP startup]}, journal = {Nature communications}, volume = {12}, year = {2021}, month = {09/2021}, pages = {1{\textendash}12}, type = {Journal Article}, abstract = {

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-β-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 {\r A} resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

}, doi = {https://doi.org/10.1038/s41467-021-25679-0}, url = {https://www.nature.com/articles/s41467-021-25679-0}, author = {Bharatham, Nagakumar and Bhowmik, Purnendu and Aoki, Maho and Okada, Ui and Sharma, Sreevalli and Yamashita, Eiki and Shanbhag, Anirudh P and Rajagopal, Sreenath and Thomas, Teby and Sarma, Maitrayee and others} } @article {764, title = {Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 May 08}, pages = {7263}, abstract = {

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-25407-7}, author = {Hameed P, Shahul and Bharatham, Nagakumar and Katagihallimath, Nainesh and Sharma, Sreevalli and Nandishaiah, Radha and Shanbhag, Anirudh P and Thomas, Teby and Narjari, Riya and Sarma, Maitrayee and Bhowmik, Purnendu and Amar, Prakruthi and Ravishankar, Rajani and Jayaraman, Ramesh and Muthan, Kubendran and Subbiah, Ramesh and Ramachandran, Vasanthi and Balasubramanian, V and Datta, Santanu} }