Study of face mutants of Hfq and their role in the pathophysiology of Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an opportunistic pathogen known to cause a multitude of infections in immunocompromised hosts. The rise in frequent multidrug-resistant (MDR) infections of A. baumannii has made this microbe a pathogen of great concern. With its highly plastic genome and an intrinsic propensity towards horizontal gene transfer, this pathogen can develop or acquire novel resistance mechanisms and survival strategies under stress conditions. Additionally, A. baumannii strains must withstand diverse stress conditions, especially in a hospital environment. As a result, this pathogen must tolerate a plethora of environmental and host-mediated stress responses to survive and establish a successful infection. Hence, the pathogenic A. baumannii must adapt swiftly to its rapidly changing environmental stressors. A. baumannii achieves the same using post-transcriptional regulation of gene expression, mediated by small RNAs and Hfq chaperone. Hfq brings about sRNA-mRNA interaction that results in robust fine-tuning of the genetic circuit. Several conserved positively charged amino acid residue on Hfq interacts with the negatively charged phosphodiester backbone of cognate RNA molecules to bring about these genetic regulations. The current project aims to decipher the role of conserved residues of Hfq and their impact on regulatory processes in this pathogen. Here we have constructed face mutants of Hfq on the genome of A. baumannii ATCC17978. These strains were further subjected to a diverse set of physiological assays like growth kinetics, biofilm formation, and MIC profiling. Next, a differential whole transcriptome and total sRNA-ome analysis was performed using the wildtype and the hfq deletion mutant strain. Further analysis of the sRNA-ome data was carried out using the CopraRNA tool to predict probable targets of these novel sRNAs and link them to the differentially expressed mRNA data. Next, total RNA was isolated from different strains of A. baumannii and assessed for the levels of a couple of sRNAs, identified in a previous study and came up as hits in the sRNA-ome data. Next, these strains were evaluated for their virulence ability in a C. elegans model. As a result of this study, we were able to generate single amino acid substitution mutants of Hfq successfully in the genome of A.baumannii ATCC17978. Our results suggest that the face mutants of Hfq showed compromised pathophysiological fitness under the tested conditions. Furthermore, using the transcriptome analysis, we found out that several sRNAs are differentially regulated by Hfq and might be subjected to dysregulation in the face mutants. Using qRT-PCR analysis, we showed that there’s a differential expression profile of sRNA in the Hfq face mutants. Finally, we found out that the face mutants are less virulent than the wild type in the C. elegans survival model. Our study found that the Hfq binding face amino acid residues are critical for A. baumannii’s pathophysiological fitness, antibiotic tolerance, and virulence. Furthermore, this has laid the groundwork for the identification of novel sRNA-mediated critical genetic circuits in this pathogen. Overall, such insight will aid in novel arget-based drug development to combat MDR strains of this pathogen in the future.