Research Projects

Acinetobacter baumannii is a major hospital-acquired pathogen with rapid multidrug resistance, making current treatments ineffective. Its adaptability is linked to post-transcriptional regulation by sRNAs and Hfq, but their roles in metabolism and virulence are unclear. This proposal aims to uncover how sRNAs like AbsR1 regulate key pathways such as arginine metabolism, and investigate for the first time the role of sRNAs in regulating cellular proteins, providing insights into new therapeutic targets against multidrug-resistant A. baumannii. Objective 1: To carry out structural and functional characterization of AbsR1 sRNA Objective 2: To investigate the regulatory roles of AbsR1 in arginine metabolism and other physiological pathways Objective 3: To understand riboregulatory roles of bacterial small non-coding RNAs This proposal hypothesizes that sRNAs, particularly AbsR1, act as key regulators of metabolism and virulence in A. baumannii via both classic post-transcriptional and novel riboregulatory mechanisms. It tests if AbsR1 directly controls arginine catabolism genes and their role in lung infection, and explores sRNA–protein interactions using pull-down and proteomics, integrating structural, metabolic, and riboregulatory approaches with collaborator Prof. Kiran Ambatipudi from IIT Roorkee. Experiments: Under objective 1- In Vitro Transcription and Radiolabeling of AbsR1, Secondary Structure Determination Using Lead Probing, Construction and Validation of AbsR1 Hfq-Binding Mutants, Electrophoretic Mobility Shift Assays for sRNA-Hfq Interactions, RNase E Degradation Assays for Stability Analysis, Population-Level Expression Analysis Using Flow-FISH. Under objective 2- Quantitative Analysis of Arginine Uptake Efficiency, Transcriptional Validation of Arginine Metabolism Genes, Pulse-Chase Analysis of AbsR1 Expression Effects, Direct Binding Validation Through EMSA and ITC, Global Target Identification Using GRIL-seq, Virulence Assessment in Murine Lung Infection Models. Under objective 3- Generation of Aptamer-Tagged sRNAs for Protein Interaction Studies, UV-Crosslinking and Proteomic Analysis of sRNA-Protein Interactions, Validation and Characterization of sRNA-Protein Binding Interactions, Functional Analysis of sRNA-Protein Interactions, Assessment of Riboregulatory Effects on Bacterial Virulence. Estimate of significance: Successful completion of this proposal will provide major advances in both basic science and clinical application. Fundamentally, it will establish the first comprehensive framework for post-transcriptional and riboregulatory networks in Acinetobacter baumannii, addressing key gaps in bacterial RNA biology. Structural and functional analysis of AbsR1 and its network will reveal new mechanisms of sRNA-mediated metabolic control, potentially redefining how bacterial adaptation is understood in pathogens. Importantly, exploring sRNA–protein interactions in bacteria, a mechanism well known in eukaryotes but unstudied in prokaryotes, could open a new field of bacterial regulation and transform our concept of RNA-mediated gene control. Clinically, the research is highly relevant, as carbapenem-resistant A. baumannii is a WHO critical priority. Identifying sRNA targets and mechanisms with GRIL-seq may yield novel therapeutic targets for RNA-based interventions, enabling precision antimicrobials that avoid the disadvantages of traditional antibiotics. Demonstrating sRNA control of arginine metabolism, essential for lung infection, will directly inform strategies against respiratory infections, the main clinical issue caused by this pathogen. Overall, RNA-based therapeutics, such as antisense oligonucleotides or small molecules targeting sRNAs, could offer new solutions as antibiotic resistance rises. Ultimately, this work bridges basic bacterial biology and clinical need, potentially leading to breakthrough therapies for a major healthcare threat.