Regulation of biofilm formation and desiccation tolerance in the critical priority pathogen Acinetobacter baumannii

Abstract

The current ’’silent pandemic’’, caused by the increasing levels of antibiotic resistance amongst pathogenic bacteria and fuelled by the lack of new antibiotics, seriously threatens global healthcare systems. Acinetobacter baumannii is the most critical pathogen, for which novel therapeutic interventions are urgently needed. It is highly prevalent in hospitals and more alarmingly in intensive care units, where it can persist on surfaces in the absence of water and nutrients for months. Additionally, the majority of the nosocomial infections caused by this pathogen are biofilm-related. Biofilm formation is a universal bacterial survival strategy that confers protection to cells from harsh environmental conditions. However, majority of the regulatory mechanisms orchestrating biofilm formation and desiccation tolerance in A. baumannii are still elusive. In this thesis we present the screening of the Manoil lab transposon mutant library for differential biofilm biomass levels. This led to uncovering CavA adenylate cyclase (AC) as a novel negative biofilm mediator. Subsequently we characterise the role of CavA as the dominant AC under the conditions tested and reveal the cAMP signalling cascade in A. baumannii AB5075 strain. We show that cAMP inhibits biofilm formation while enhancing motility, which is exerted via its effector protein Vfr, and inhibits quorum sensing (QS). In addition to QS, we elucidate the hierarchy of signalling cascades that cAMP orchestrates, which also involves the other major second messenger c-di-GMP. We demonstrate that contrary to the current dogma, in A. baumannii cAMP increases intracellular levels of c-di-GMP via transcriptional regulation of DgcC diguanylate cyclase (DGC) and PdeE phosphodiesterase (PDE). Furthermore, we unveil that cAMP modulates virulence and transient antibiotic resistance. We also present that AB5075 strain possesses an additional AC, CavB, and a second potential cAMP effector protein Crp. In addition, we investigate regulators governing A. baumannii desiccation tolerance. We show that A. baumannii needs low cAMP levels, via CavB AC downregulation and CpdA PDE upregulation, while c-di-GMP related DGC DgcB negatively impacts desiccation tolerance. Moreover, the protective function of A. baumannii type VI secretion system under desiccation is uncovered. Finally, we demonstrate the central role of phenylacetic acid (PAA) catabolism in the desiccation tolerance of this pathogen. Our data shows that accumulation of intracellular PAA drives A. baumannii cells to enter a viable but non-culturable (VBNC) state, which is a persister strategy used by bacteria to withstand unfavourable conditions. These findings uncover central regulators of fundamental A. baumannii behaviours (formation of biofilms, tolerance to desiccation and VBNC state), which facilitate the survival of this pathogen in patients and hospitals. These new insights uncover targets for the development of novel therapeutics and disinfectants against A. baumannii and will contribute to improved detection and decontamination practices in the future.