Periodic Reporting for period 1 - ERA (Epigenetic Regulation in Acinetobacter baumannii)
Reporting period: 2020-07-01 to 2022-06-30
What is the problem/issue being addressed?
Bacterial gene expression regulation allows bacteria to adapt to environmental changes. The primary and most well-studied mechanism of gene expression regulation is the interaction of specific proteins, called transcription factors, with DNA. DNA methylation can alter the affinity of transcription factors to their target sites, and, as a result, modulate gene expression. The impact of DNA methylation is global and affects different aspects of bacterial physiology. However, the functional mechanisms of the methylation effect on gene expression are unclear. DNA is methylated by specific proteins, called DNA methyltransferases. These proteins can be a part of bacterial Restriction-Modification (R-M) systems where they are used to differentiate methylated host DNA from unmethylated foreign DNA. In this project, ERA, the Fellow investigated the role of methylation in Acinetobacter baumannii. The understanding of regulatory networks in these bacteria is essential for the development of new drugs and the understanding of the mechanisms of antibiotic resistance.
Why is it important for society?
The project's results contribute to studying the bacterial pathogen A. baumannii. In 2017, the World Health Organisation included these bacteria in the critical priority list of pathogens for new drug development. The results improved our knowledge of epigenetic regulation mechanisms in bacteria and the diversity of their defence systems. The obtained results can reveal potential new drug targets and achieve healthy lives for all people which is one of the UN Sustainable Development Goals.
The project was carried out at the Microbiology Department of Trinity College Dublin, so some tasks were done as student projects. The solution to fundamental scientific problems is an essential part of student education.
What are the overall objectives?
The project included the following objectives, organised in Work Packages (WP).
Objective 1 (WP1). Bioinformatic analysis of the diversity and distribution of R-M systems and orphan MTases in known A. baumannii genomes to reveal regulatory DNA methyltransferase.
Objective 2 (WP2). Establishing the role of the most widespread MTases in global gene expression, fitness, and virulence in experimental fitness and antibiotic-resistance assays, and infection models.
Objective 3 (WP3). Determination of the direct and indirect regulatory role of the MTases by experimental and bioinformatic methods.
The project also includes WP4, Training, and WP5 - Data dissemination.
In the project, the Fellow employed a powerful combination of whole-genomic and transcriptomic approaches and phenotypic assays to characterise the role of methylation in A. baumannii. The project revealed the effect of methylation on gene expression and pathogenic traits of the bacteria, including biofilm formation, and virulence.
Bacterial gene expression regulation allows bacteria to adapt to environmental changes. The primary and most well-studied mechanism of gene expression regulation is the interaction of specific proteins, called transcription factors, with DNA. DNA methylation can alter the affinity of transcription factors to their target sites, and, as a result, modulate gene expression. The impact of DNA methylation is global and affects different aspects of bacterial physiology. However, the functional mechanisms of the methylation effect on gene expression are unclear. DNA is methylated by specific proteins, called DNA methyltransferases. These proteins can be a part of bacterial Restriction-Modification (R-M) systems where they are used to differentiate methylated host DNA from unmethylated foreign DNA. In this project, ERA, the Fellow investigated the role of methylation in Acinetobacter baumannii. The understanding of regulatory networks in these bacteria is essential for the development of new drugs and the understanding of the mechanisms of antibiotic resistance.
Why is it important for society?
The project's results contribute to studying the bacterial pathogen A. baumannii. In 2017, the World Health Organisation included these bacteria in the critical priority list of pathogens for new drug development. The results improved our knowledge of epigenetic regulation mechanisms in bacteria and the diversity of their defence systems. The obtained results can reveal potential new drug targets and achieve healthy lives for all people which is one of the UN Sustainable Development Goals.
The project was carried out at the Microbiology Department of Trinity College Dublin, so some tasks were done as student projects. The solution to fundamental scientific problems is an essential part of student education.
What are the overall objectives?
The project included the following objectives, organised in Work Packages (WP).
Objective 1 (WP1). Bioinformatic analysis of the diversity and distribution of R-M systems and orphan MTases in known A. baumannii genomes to reveal regulatory DNA methyltransferase.
Objective 2 (WP2). Establishing the role of the most widespread MTases in global gene expression, fitness, and virulence in experimental fitness and antibiotic-resistance assays, and infection models.
Objective 3 (WP3). Determination of the direct and indirect regulatory role of the MTases by experimental and bioinformatic methods.
The project also includes WP4, Training, and WP5 - Data dissemination.
In the project, the Fellow employed a powerful combination of whole-genomic and transcriptomic approaches and phenotypic assays to characterise the role of methylation in A. baumannii. The project revealed the effect of methylation on gene expression and pathogenic traits of the bacteria, including biofilm formation, and virulence.
At the beginning of the project, (WP1) the Fellow identified DNA methyltransferases in ~ 5000 available complete genomes of A. baumannii. About 32000 predicted R-M-related proteins were grouped in 1055 groups by sequence similarity (% identity > 95%). Only one DNA methyltransferase was encoded in all analysed strains. This methyltransferase was chosen for further experimental study.
Then the Fellow created the methyltransferase deletion mutant for the A. baumannii AB5075 strain (WP2). Transcriptomic data analysis showed 496 differentially expressed genes between the methyltransferase deletion mutant and wild type. To charachterise the role of methylation, the Fellow performed several phenotypic assays, including motility test, antibiotic sensitivity test, biofilm formation, growth, infection of Galleria mellonella larvae, and eukaryotic cells for methyl-deficient mutant and wild-type strains. The mutant had several important phenotypic traits, including differences in growth, plastic surface attachment, biofilm formation, and motility.
We found 273 out of 496 differentially expressed genes have the RAATTY site in the upstream region (WP3). It can explain the difference in the expression of these genes in the mutant strain. We identified several potentially methylation-sensitive transcription factors.
During this project, the Fellow got training (WP4) in bioinformatics, molecular microbiology, RNA-seq data analysis, and infection models at Trinity College Dublin and University of Lyon. Additionally, the Fellow took courses on available at TCD. To facilitate knowledge transfer the Fellow co-supervised 3 BSc and 1 MSc diploma projects, focused on the study of A. baumannii at Trinity College Dublin. The MSc diploma results on characteristics of a rare species of Acinetobacter soli AS15, isolated from an Irish Hospital, were published recently. The new skills will help the Fellow to to facilitate her career prospects.
Results of the project were presented (WP5) at two conferences, ISMB/ECCB 2021 (online, ~2000 participants), AcinetoVibes 2022 (online, ~ 100 participants). Results of the ERA project will be reported in forthcoming papers on the epigenetic regulation of A. baumannii. Results of the project were presented to the general public during the European Researchers’ Night 2021.
The conclusion of the action
Scientific objectives of the project are achieved. We found that the AbaM deletion mutant has impaired growth, plastic attachment, and biofilm formation and is less virulent. We identified differentially expressed genes that cause the abaM deletion mutant phenotype.
Training objectives are achieved. The Fellow improved her skills in Data Analysis and Genomic/Transcriptomyc data handlyng, obtained new wet-lab skills. Additionally, the Fellow got transferrable skills, such as Project Management and Scientific Communication. The obtained skills and results will help the Fellow to establish herself as an independent investigator in the field of epigenomics and gene expression regulation.
Then the Fellow created the methyltransferase deletion mutant for the A. baumannii AB5075 strain (WP2). Transcriptomic data analysis showed 496 differentially expressed genes between the methyltransferase deletion mutant and wild type. To charachterise the role of methylation, the Fellow performed several phenotypic assays, including motility test, antibiotic sensitivity test, biofilm formation, growth, infection of Galleria mellonella larvae, and eukaryotic cells for methyl-deficient mutant and wild-type strains. The mutant had several important phenotypic traits, including differences in growth, plastic surface attachment, biofilm formation, and motility.
We found 273 out of 496 differentially expressed genes have the RAATTY site in the upstream region (WP3). It can explain the difference in the expression of these genes in the mutant strain. We identified several potentially methylation-sensitive transcription factors.
During this project, the Fellow got training (WP4) in bioinformatics, molecular microbiology, RNA-seq data analysis, and infection models at Trinity College Dublin and University of Lyon. Additionally, the Fellow took courses on available at TCD. To facilitate knowledge transfer the Fellow co-supervised 3 BSc and 1 MSc diploma projects, focused on the study of A. baumannii at Trinity College Dublin. The MSc diploma results on characteristics of a rare species of Acinetobacter soli AS15, isolated from an Irish Hospital, were published recently. The new skills will help the Fellow to to facilitate her career prospects.
Results of the project were presented (WP5) at two conferences, ISMB/ECCB 2021 (online, ~2000 participants), AcinetoVibes 2022 (online, ~ 100 participants). Results of the ERA project will be reported in forthcoming papers on the epigenetic regulation of A. baumannii. Results of the project were presented to the general public during the European Researchers’ Night 2021.
The conclusion of the action
Scientific objectives of the project are achieved. We found that the AbaM deletion mutant has impaired growth, plastic attachment, and biofilm formation and is less virulent. We identified differentially expressed genes that cause the abaM deletion mutant phenotype.
Training objectives are achieved. The Fellow improved her skills in Data Analysis and Genomic/Transcriptomyc data handlyng, obtained new wet-lab skills. Additionally, the Fellow got transferrable skills, such as Project Management and Scientific Communication. The obtained skills and results will help the Fellow to establish herself as an independent investigator in the field of epigenomics and gene expression regulation.
The ERA project showed the importance of RAATTY methylation for the important phenotypic traits of A. baumannii, including cell growth, motility, biofilm formation, and virulence. We created a methyltransferase deficient mutant and found 426 genes differentially expressed between the parental strain and the mutant. These genes can be directly or indirectly regulated by methylation. The importance of this project is due to the clinical significance of A. baumannii, its resistance to modern antibiotics and its ability to persist in hospitals. Results of the project are essential to further explore epigenetic mechanisms and regulatory MTases as possible targets for drug development. The publication of the results will improve the position of Ireland and TCD in molecular microbiology research. Dr. Carsten Kröger will use the results to plan his further studies of A. baumannii and in his future collaborations with other colleagues from Europe. The Fellow and Dr. Carsten Kröger plan future collaborations in epigenetic regulations and genomic studies.