Browsing by Author "Cheng, Hsin-Yao"

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Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43
(2010-07-24)
Cheng, Hsin-Yao
;
Chen, Yi-Fong
;
Peng, Hwei-Ling
;
生物科技學系
;
分子醫學與生物工程研究所
Background: The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria. Methods: In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen Klebsiella pneumoniae were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of pmr genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and in vitro phosphor-transfer assay. Results: Deletion of the rcsB, which encoded an activator for the production of capsular polysaccharide, had a minor effect on K. pneumoniae resistance to polymyxin B. On the other hand, deletion of ugd or pmrF gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in Salmonella, expression of pmrD in K. pneumoniae was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of pmr for the resistance to polymyxin B. Conclusions: The study reports a role of the capsular polysaccharide level and the pmr genes for K. pneumoniae resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of pmr expression was demonstrated. In comparison to the pmr regulation in Salmonella, PhoP in K. pneumoniae plays a major regulatory role in polymyxin B resistance.
Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae
(2010-12-01)
Luo, Shih-Chi
;
Lou, Yuan-Chao
;
Cheng, Hsin-Yao
;
Pan, Yun-Ru
;
Peng, Hwei-Ling
In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D) = 1.74 +/- 0.81 mu M vs. K(D) = 236 +/- 48 mu M). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six beta-strands and a flexible C-terminal alpha-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the beta-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52,Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS. (C) 2010 Elsevier Inc. All rights reserved.