Characterising the small RNA-regulatory architecture of Acinetobacter baumannii

Abstract

The Gram-negative bacterium Acinetobacter baumannii is a significant opportunistic pathogen in critically ill and hospitalised patients. It exhibits a remarkable ability to survive under harsh environmental conditions, resisting desiccation, oxidative stress, and exposure to disinfectants. Clinical isolates frequently form biofilms and employ micronutrient acquisition strategies, allowing colonisation of surfaces in clinical settings and facilitating propagation within hosts. Moreover, many A. baumannii strains are acquiring multidrug resistance (MDR), limiting the efficacy of antimicrobial treatments and underscoring the urgent need to understand the genetic regulators underpinning its environmental persistence and MDR mechanisms. While much attention has been given to transcriptional regulators, the role of bacterial small RNAs (sRNAs) in A. baumannii remains poorly characterised. sRNAs are known to regulate gene expression in other bacteria through direct, imperfect antisense base-pairing. This study sought to elucidate A. baumannii sRNA interactions by identifying their RNA partners using Hi-GRIL-seq, a high-throughput RNA proximity ligation and sequencing technique applied under several experimental conditions. We identified forty sRNAs forming sRNA-RNA chimeras, suggesting their involvement in gene regulation in A. baumannii. One sRNA, Aar, was found to regulate four mRNA targets, including the outer membrane protein CarO. Aar binds the translational initiation regions of these targets via a conserved seed region, as demonstrated through both in vitro and in vivo approaches. This binding inhibits translation without inducing mRNA degradation. Overexpression of Aar led to reduced CarO levels, highlighting its role in fine-tuning outer membrane protein expression. Another sRNA, Arp, was found to regulate the major type IV pilin subunit PilA, crucial for DNA uptake in A. baumannii. Arp sequesters the translational initiation region of pilA mRNA, preventing ribosome binding in a heterologous reporter system. While further work is needed to fully understand Arp's function, it may shed light on the mechanisms controlling horizontal gene transfer in this pathogen. This study provides the first mechanistic insights into post-transcriptional regulation in A. baumannii and serves as a valuable resource for future RNA-centred investigations in this organism.