title: Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 reveals a tight link between tyrosine phosphorylation and virulence.
abstract: Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD(50) increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD(50) increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.
authors.affiliation: Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Miao-Hsia
authors.initials: MH
authors.lastname: Lin
authors.forename: Tung-Li
authors.initials: TL
authors.lastname: Hsu
authors.forename: Shu-Yu
authors.initials: SY
authors.lastname: Lin
authors.forename: Yi-Jiun
authors.initials: YJ
authors.lastname: Pan
authors.forename: Jia-Tsrong
authors.initials: JT
authors.lastname: Jan
authors.forename: Jin-Town
authors.initials: JT
authors.lastname: Wang
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.forename: Shih-Hsiung
authors.initials: SH
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016667:Bacterial Capsules / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015169:Colony Count, Microbial; D007711:Klebsiella pneumoniae / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D040901:Proteomics; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D019000:Phosphotyrosine
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