Abstract
Pseudomonas syringae is a Gram-negative pathogen infecting many plants, including economically valuable crops, resulting in substantial annual losses worldwide. P. syringae is equipped with multiple virulence-related pathways, including type III secretion system (T3SS), motility, and biofilm formation. T3SS is the primary weapon for P. syringae to cause severe disease, which is highly induced by nutrient-deficient minimal medium (MM) or in planta and controlled by a cluster of transcription factors (TFs) or two-component systems (TCSs). However, except for the regulatory elements, the roles of epigenetics and RNA structure alteration in the pathogenesis of P. syringae are still unknown. Therefore, we used different sequencing technologies to fill these gaps.Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. RNA secondary structure can be visualised at the single-nucleotide level using chemical probing like Dimethyl sulfate (DMS). DMS can methylate unpaired adenine (A) and cytosine (C), identified by interrupting reverse transcription, followed by deep sequencing and bioinformatic analysis to unveil diverse RNA structures. We employed DMS sequencing (DMS-seq) to reveal that the mRNA structure changes substantially in MM and tunes global translation efficiency (TE), thereby inducing virulence. In this study, we identified that Hfq, an RNA binding protein, modulated RNA structure, with its distal surface assuming a pivotal role in overseeing the dynamic changes in RNA structure and the expression of virulence factors. DNA pull-down assays determined that the sigma factor RpoS and the transcription factor CysB governed hfq expression in P. syringae following MM induction. Overall, our study unveiled that under MM conditions, RpoS and CysB jointly orchestrate dynamic shifts in RNA structure through the RNA-binding protein Hfq, presenting a potential mechanism in other microorganisms.
Bacterial pathogens, such as P. syringae, leverage epigenetic mechanisms, including DNA methylation, to adapt to environmental changes, playing crucial roles in various biological processes. Through the application of single-molecule real-time sequencing (SMRT-seq), this study profiles the DNA methylation landscape across model pathovars of P. syringae, uncovering a conserved Type-I restriction-modification system (HsdMSR) associated with N6-methyladenine (6mA) catalyzation. Subsequent transcriptomic analysis highlighted the involvement of HsdMSR in virulent and metabolic pathways, including the T3SS, biofilm formation, and TE. The regulatory effect of HsdMSR on transcription was dependent on both strands being fully 6mA methylated. Overall, this work illustrated the methylation profile in P. syringae and the critical involvement of DNA methylation in regulating virulence and metabolism. Thus, this work contributes to a deeper understanding of epigenetic transcriptional control in P. syringae and related bacteria.
The gram-negative bacterium Pseudomonas aeruginosa is a major human opportunistic pathogen that grows ubiquitously. P. aeruginosa tends to cause infection in burn victims, patients with cystic fibrosis (CF), and individuals hospitalised for long periods. It utilises versatile pathways to exert its virulence, such as biofilm formation, quorum sensing (QS), T3SS, T6SS, motility, and antibiotic resistance, all under the control of a complicated TF regulation network (TRN). Despite the TRN being essential for understanding how bacterial genes are regulated synergistically, almost half of the TFs in P. aeruginosa remain annotated with biological functions, and their downstream targets and upstream regulators remain unknown. To fill this knowledge gap, our research extended to employ chromatin immunoprecipitation sequencing (ChIP-seq) to map the binding sites of 172 transcription factors in the PAO1 strain. By assembling a hierarchical network and identifying key regulatory motifs, we unveil the complex interplay among transcription factors that govern virulence pathways. The establishment of a web-based database consolidating this data alongside high-throughput techniques offers valuable insights into bacterial pathogenesis, laying the groundwork for future therapeutic interventions.
In summary, this thesis presents an integrated analysis of the epigenetic and RNA structure alteration that underlie the adaptability and pathogenicity of P. syringae. By mapping the methylome and transcriptome of this bacterium, this work contributes significantly to our understanding of bacterial epigenetics and post-transcriptional regulation. We also elucidated the regulatory networks that govern the behavior of P. aeruginosa and identified novel master regulators involved in virulence. The insights garnered from this research not only enhance our knowledge of Pseudomonas but also have broad implications for the study of bacterial pathogenesis, offering new avenues for the development of antimicrobial strategies.
| Date of Award | 27 Aug 2024 |
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| Original language | English |
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| Supervisor | Xin DENG (Supervisor) & Yung Fu CHANG (External Co-Supervisor) |