SACM - Ireland

Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9656

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The transcriptional landscape and small RNAs in Acinetobacter baumannii AB5075 during antibiotic and environmental stress
(University of Dublin, 2024-06-28) Bakheet, Ali Ahmad; Kröger, Carsten
Multi-drug resistant Acinetobacter baumannii is considered a major challenge because it causes a variety of serious infections. Studying these bacteria at the level of their genetic response could facilitate finding a way to treat them. Tracking gene expression throughout the growth phases or when affected by any environmental stresses can deepen our understanding of how genes work and how cells function. Here, we studied global gene expression of A. baumannii AB5075 using RNA-seq under the influence of 12 different stresses divided into three groups. The first group includes five growth stages in rich medium, the second group includes four environmental shocks (osmotic, iron limitation and two antibiotic shocks), and the third group contains temperatures shift experiments (between 22°C and 37°C). An important finding is the dramatic up-regulation of the phenyl-acetic acid (paa) pathway including paaA, paaB, paaC, paaJ, paaK and paaZ in late exponential and early stationary phase in rich medium which influences virulence, antibiotic resistance, and biofilm formation. In the environmental shock conditions, we reveal that the transcriptional responses were similar between osmotic shock and iron limiting conditions, and the effect of antibiotics (exposure to chloramphenicol and tetracycline), respectively, indicating that similar pathways are involved in these stress responses. These included genes encoding siderophore Acinetobactin encoding genes that were up-regulated in both osmotic shock and iron limiting conditions. The two antibiotic shocks showed gene expression changes to a much larger than osmotic shock and iron limiting conditions suggesting that protein synthesis stress require a much larger transcriptomic response to cope with the stress compared to the osmotic and iron limitation stress. A response to the antibiotic stress was observed to be a down-regulation of the NADH-quinone oxidoreductase complex, a central protein complex in the respiratory chain, indicating the antibiotics could influence the proton gradient across the membrane or that further growth and ATP synthesis should be immediately stopped. When growing at different temperatures, there were large changes to the transcriptome in all temperature comparisons. The virulence-associated otsAB operon and paa genes were strongly up-regulated upon temperature shift. Type IV pilus encoding genes were down-regulated genes when cells were experiencing a temperature upshift (TS) which could mean that DNA uptake is at least transiently down-regulated when A. baumannii enters a warm-blooded host. In contrast, a temperature downshift induced expression of the Type 6 Secretion System and Csu pili. We also provide a first expression landscape of sRNAs in the suite of 12 conditions. We observed dynamic expression of many small RNAs across the 12 conditions. The sRNAs sRNA99 and sRNA100 very quickly dropped in abundance when the cells experienced antibiotic shocks which was independently validated by Northern blotting for sRNA99 and sRNA100. Deletion of sRNA99 and sRNA100, resulted in large expression changes in MEP in the mutants compared to the WT, which will require further analysis. The sRNA44 was investigated mechanistically in more detail in this study because previous data in A. baumannii AB5075 suggested that sRNA44 was ligated to the mRNA of the biofilm-associated bap gene. We show that sRNA44 translationally represses bap translation in E. coli and that overexpression of sRNA44 reduces bap mRNA levels, likely induced by a base-pairing mechanism. Deletion of sRNA44 and bap showed that bap is required for full biofilm formation after 24h, while deletion of sRNA44 increased biofilm formation suggesting that sRNA44 is a regulator of A. baumannii biofilm formation.
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Transcriptomic and Functional Analysis of Two-Component Systems AdeRS, BaeRS, GacAS in Acinetobacter baumannii AB5075
(Saudi Digital Library, 2025) Sulimani, Maha; Kröger, Carsten
Antimicrobial resistance constitutes one of the most significant global health challenges of our era, with Acinetobacter baumannii becoming a key priority pathogen owing to its extraordinary capacity to withstand various classes of antibiotics. A primary factor contributing to this resilience is the function of regulatory proteins, especially Two-Component Systems (TCSs), which allow the bacterium to detect environmental cues and swiftly modify gene expression. Nevertheless, the overarching regulatory networks of these systems are still not well comprehended. The objective of this thesis was to examine the regulatory functions of specific two-component systems (TCSs) AdeRS, BaeRS, and GacSA in A. baumannii AB5075, as well as to investigate their roles in antimicrobial resistance and various adaptive phenotypes. Through transcriptomic analysis (RNA-seq), I detected genome wide expression alterations in mutant strains that lacked these regulators when compared to the wild-type during exponential and stationary growth phases. Phenotypic assays, which included minimum inhibitory concentration (MIC) determination, disk diffusion, phenotypic microarrays and growth assays, were conducted to establish a correlation between transcriptional changes and antibiotic susceptibility and metabolism. In order to delve deeper into gene regulation, transcriptional start sites (TSSs) were delineated through differential RNA-seq, which facilitated the identification of promoter motifs and offered insights into the structure of transcriptional regulation in A. baumannii. Moreover, chromatin immunoprecipitation coupled with sequencing (ChIP-seq) was utilized to comprehensively map the binding sites of essential transcription factors (AdeR, BaeR, and GacA), uncovering their direct regulons and preferences for DNA binding. The results outlined in this thesis enhance our comprehension of TCS mediated regulation in A. baumannii. They reveal that AdeRS, BaeRS, and GacSA are responsible not only for the regulation of antibiotic resistance factors but also for affecting wider physiological mechanisms that aid in bacterial survival and adaptation. A comparative transcriptomic analysis of the adeRS, baeRS, and gacSA two-component systems has unveiled distinct regulatory effects on gene expression in Acinetobacter baumannii AB5075. Both adeR and adeS mutants exhibited numerous transcriptional alterations, particularly a downregulation of the adeABC efflux pump genes, thereby confirming their involvement in the regulation of antibiotic resistance. In the baeRS system, baeS demonstrated a more extensive transcriptomic influence compared to baeR, especially under the Late Stationary Phase LSP condition, where baeR was found to regulate only two genes. The gacA and gacS mutants revealed the highest number of differentially expressed genes across all systems, with gacA notably regulating the small RNAs sRNA100 and sRNA99, as indicated by peaks observed in chip seq data. The phenotypic characterization conducted through Kirby–Bauer disk diffusion assays indicated that all adeRS, baeRS, and gacSA mutants displayed measurable inhibition zones against specific antibiotics, implying partial changes in their susceptibility profiles. Nevertheless, minimum inhibitory concentration (MIC) testing revealed that these mutants continued to exhibit resistance, suggesting that although the regulatory disruptions influenced gene expression, they were not adequate to completely undermine the antibiotic resistance phenotypes.
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