GSE202420
DNA Methyltransferase regulates nitric oxide homeostasis and virulence in a chronically adapted Pseudomonas aeruginosa strain
Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively. LC-MS/MS analysis of DNA methylation was employed to verify the MTase activities. Transcriptomic analysis showed that ?M.PaeTBCFII knockout mutant significantly downregulated nitric oxide reductase (NOR) regulating and coding gene expression such as nosR and norB, which contain methylated motifs in their promoters or coding regions. These predicted target motifs in nosR and norB were not methylated in the ?M.PaeTBCFII knockout mutant. ?M.PaeTBCFII exhibited reduced intercellular survival capacity in NO-producing RAW 264.7 macrophages and attenuated virulence in Galleria mellonella infection model. While the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII is frequently exists in P. aeruginosa sp as well as other bacteria species. Our work therefore provided new insights on the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa. Overall design: P. aeruginosa TBCF were grown in Luria-Bertani (LB) liquid media at 37°C with constant shaking at 220 rpm with aeration. The in-frame deletion mutagenesis of methyltransferase ap or/and cp was performed through two-step allelic exchange. P. aeruginosa TBCF and mutants were cultured to mid log phase. Total RNA extraction and purification was performed for 3 biological replicates of each strain using RNeasy Mini Kit (QIAGEN) and RNase-Free DNase Set (QIAGEN) according to the manufacturer's protocol. 1µg of total RNA was used for the construction of sequencing libraries. RNA sequencing was performed on Illumina NovaSeq 6000 sequencing platform generating 150 bp paired-end reads. Raw reads of the samples were preprocessed and analyzed using RSEM v1.3.1 with unique mapping setting (--bowtie-m), using P. aeruginosa TBCF genome as reference.