Characterization and substrate identification of LMW-PTP Wzb in Klebsiella pneumoniae NTUH-K2044

Liver abscess with metastatic complications caused by Klebsiella pneumoniae (K. pneumoniae) is an emerging infectious disease in Taiwan in the recent years. The human bacterial pathogen was wrapped in a physical barrier of exopolysaccharides. The sugar coated structure, termed capsular polysaccharides (CPS), is an important virulence factor which protects the pathogen from attack by the host immune system. It has been documented that a cassette of genes involved in CPS development are gathered at the capsule biosynthesis locus (cps). The Wzb gene is localized within such gene cluster which determines synthesis and assembly of CPS. It has been suggested that wzb is involved in the regulation of CPS biosynthesis, since the capsule is eliminated in the wzb knockout strain. Furthermore, Wzb is also considered as a kind of low molecular weight protein tyrosine phosphatases (LMW-PTP) based on the protein sequence alignment. n order to confirm the role of Wzb in regulating bacterial CPS biosynthesis through protein tyrosine phosphorylation, we first cloned the Wzb from the liver abscess strain K. pneumoniae NTUH-K2044 and tagged the protein with both histidines (His) and haemagglutinin epitop (HA) at the C-terminus. Meanwhile, substrate-trapping mutants, C9S, D115A and C9S/D115A, were also generated based on the information of the active site. The maximal enzymatic activity of Wzb was achieved at a pH of 5.5 and the corresponding kinetic constants Km, Vmax, Kcat and Kcat/Km, measured at 26.8°C, were 1.35 mM, 34.8 μmole min-1 mg-1, 641.1 min-1and 475 mM-1 min-1, respectively. The inhibition assay revealed that Wzb is not inhibited by NaF and EDTA, even at a high concentration (10 mM). On the contrary, pre-incubation with 10 mM H2O2 or 1 μM vanadate or 1 mM indoacetamide caused the phosphatase to lose its activity completely. These results confirmed that Wzb of K. pneumoniae NTUH-K2044 can be classified as a LMW-PTP. n vitro dephosphorylation assay indicated that several endogenous tyrosine phosphorylated proteins can be dephosphorylated by Wzb, suggesting that these unknown proteins are potential targets of Wzb and governed by it. On the other word, the cellular activity regulated by tyrosine phosphorylation system in bacteria can be clarified by substrate identification. In order to uncover the endogenous substrates of Wzb, we perform substrate-trapping experiments to pull them out where the LMW-PTP is first adopted. Against all expectations, substrate-trapping analysis falls to identify any endogenous tyrosine phosphorylated proteins, however the tyrosine-autokinase, Wzc, can serve as a substrate of Wzb that was verified by a straightforward manner. There are still several obstacles to overcome in the recognition of natural substrates. For further understanding of the precise roles of tyrosine phosphorylation system in bacterial CPS biosynthesis or other associated cellular responses, more precise experimental methods must be exploited in the future to answer those questions. Our investigation on the substrate identification is still ongoing.