title: DNA adenine methylation modulates pathogenicity of Klebsiella pneumoniae genotype K1.
abstract: OBJECTIVE
  Klebsiella pneumoniae genotype K1 is a highly virulent pathogen that causes liver abscess and metastatic endophthalmitis/meningitis. Whether its pathogenicity is controlled by DNA adenine methylase (Dam), an epigenetic regulator of bacterial virulence gene expression, is yet unknown. We aimed to study the role of DNA adenine methylation in the pathogenicity of K. pneumoniae genotype K1.
  
  
  METHODS
  We identified the dam gene in the prototype tissue-invasive strain (NTUH-K2044) of K. pneumoniae genotype K1, using the strain's complete genome sequence in GenBank. We constructed a dam-mutant and compared it with the wild type, in terms of in vitro serum resistance and in vivo BALB/cByl mice inoculation.
  
  
  RESULTS
  Loss of Dam activity in the mutant was verified by MboI restriction digestion of the genomic DNA and a 1000-fold increase in spontaneous mutation rate. The dam mutant lost at least 68% of serum resistance when compared with the wild type (survival ratio at 1 hour: 2.6 ± 0.4 vs. 8.2 ± 1.9; at 2 hours: 3.9 ± 1.6 vs. 17.4 ± 3.6; p values < 0.05). Likewise, virulence to mice decreased by 40-fold in an intraperitoneal injection model [lethal dose, 50% (LD50): 2 × 103colony-forming units (CFUs) vs. 5 × 101CFUs] and by sixfold in a gastric ingestion model (LD50: 3 × 104CFUs vs. 5 × 103CFUs). Attenuation of the dam mutant was not attributable to its growth rate, which was similar to that of the wild type.
  
  
  CONCLUSIONS
  Our results support the view that DNA adenine methylation plays an important role in modulating the pathogenicity of K. pneumoniae genotype K1. The incomplete attenuation indicates the existence of other regulatory factors.
authors.affiliation: Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan; Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan. Electronic address: fangct@ntu.edu.tw.
authors.forename: Chi-Tai
authors.initials: CT
authors.lastname: Fang
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Yi
authors.affiliation: Department of Forensic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Chia-Tung
authors.initials: CT
authors.lastname: Shun
authors.affiliation: Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan.
authors.forename: Shih-Feng
authors.initials: SF
authors.lastname: Tsai
mesh_terms: D000225:Adenine / Q000378:metabolism*; D000818:Animals; D001770:Blood Bactericidal Activity; D019175:DNA Methylation; D004195:Disease Models, Animal; D044127:Epigenesis, Genetic; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005838:Genotype; D007710:Klebsiella Infections / Q000382:microbiology / Q000473:pathology*; D007711:Klebsiella pneumoniae / Q000145:classification / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D007928:Lethal Dose 50; D008297:Male; D008807:Mice, Inbred BALB C; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism; D014774:Virulence
chemical_list: C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine
keywords: DNA adenine methylation; Klebsiella pneumoniae; bacterial pathogenesis; genotype K1; virulence regulation