Author: Felise Adams
Adams, Felise, 2020 Biological insights into the mechanisms underpinning the pathogenic success of Acinetobacter baumannii, Flinders University, College of Science and Engineering
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Acinetobacter baumannii is a Gram-negative human pathogen that is the causative agent of several life-threatening disease states in susceptible individuals. A range of persistence and resistance mechanisms contribute to the success of this bacterium, including an ability to sense and respond to rapidly changing environments mediated by the concerted action of regulatory proteins. One key regulatory strategy A. baumannii employ is through two component signal transduction systems (TCSs). TCSs consist of two modular multi-domain proteins; a membrane-bound histidine kinase (HK) and cytosolic response regulator (RR). The HK senses alterations in the extracellular milieu, relays the signal to the RR, triggering appropriate cellular modifcations. Despite regulating a plethora of virulent phenotypes in other bacterial pathogens, the role of TCSs encoded by A. baumannii remains largely ill defined. The broad aim of this study was to examine the relative role of three distinct TCS in regulating virulence-associated strategies employed by A. baumannii.
The AdeRS TCS is known to regulate the AdeABC tripartite efflux pump in a number of clinical A. baumannii isolates. Deletion derivatives targeting adeRS, adeAB, adeA and adeB genes were generated in the well-characterised A. baumannii ATCC 17978 strain. Antibiogram analyses revealed that the genetically modified strains displayed decreased resistance to a subset of dicationic compounds compared to wildtype (WT). Trans-complementation of these mutants partially or fully restored resistance back to WT levels. The global transcriptional landscape of the ∆adeRS derivative was determined using RNA-sequencing, and revealed significant variation in expression levels of over 290 genes compared to WT. Transcriptional analyses after shock experiments with pentamidine, a newly identified intrinsic substrate, confirmed AdeRS was directly responsible for activating expression of adeAB genes. Alternate resistance mechanisms against pentamidine were further explored, revealing that resistance levels were influenced by the presence of cations and different carbon sources in the growth media.
The second TCS analysed was the putative TCS encoded at the ACX60_11155/60 loci (11155/60). This system was chosen for further investigation due to its absence in the avirulent A. baumannii SDF strain, and thus was hypothesised to regulate virulence-associated genes. In silico analyses revealed a unique architecture for the 11160 hybrid HK, categorising the protein into an uncommon family. Construction and analysis of the ∆11155 derivative in ATCC 17978 identified an autonomous insertion sequence that disrupted the function of a distal transcriptional regulator. Regeneration of ∆11155 utilising a different gene deletion method also harboured a loss-of-function mutation in the same transcriptional regulator, revealing a novel regulatory network. Broader level investigations identified that the TCS was conserved across various bacterial species from the Proteobacteria phyla, co-localising with a transport gene cluster.
In other Gram-negative pathogens, the QseBC TCS is involved in modulating an array of different virulence-associated mechansms. A homologue of this system and its proposed target, a putative signal peptide (ygiW), are present in A. baumannii; this system was chosen for further analysis. Independent deletion derivatives targeting qseBC and ygiW genes were generated in ATCC 17978 and phenotypically analysed. Fortuitously, desiccation stress analyses of ∆qseBC and ∆ygiW derivatives compared to WT led to the identification of a novel trans-activated miniature inverted-repeat transposable element (MITE), disrupting function of a known global regulator of A. baumannii. Further characterisation of the element identified its presence amongst numerous strains across the Moraexellaeae family. The unique element showed potential to influence host gene expression, identifying a unique mechanism of ‘mobile’ regulation.
Overall, studies performed on multiple TCS have identified how some pathogenic traits are regulated in A. baumannii. The work also highlights how stressors can lead to unexpected changes in the genome, as represented by movement of known and novel mobile genetic elements. As we enter the post-antibiotic era, knowledge of the regulatory mechanisms responsible for virulence factor expression could prove to be invaluable, as proteins such as TCS offer an alternative target for the treatment of infections caused by this formidable pathogen.
Keywords: Acinetobacter baumannii, bacterial regulation, mobile genetic elements
Subject: Biology thesis
Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Prof Melissa H Brown