Crystal structure and functional analysis of the Clostridium difficile sortase

  • 尹 睿婕

Student thesis: Master's Thesis

Abstract

Clostridium difficile (C difficile) is a Gram-positive spore-forming and multidrug-resistant bacterial pathogen C difficile infection mostly occurs in hospitalized patients treated with antibiotic therapies Antibiotic treatments damage host beneficial bacteria allowed C difficile has chance to proliferate and exotoxin secretion result in Clostridium difficile-associated disease (CDAD) Recently increasing numbers of drug-resistant and highly pathogenic strains of C difficile were found due to the administration of broad-spectrum antibiotics Therefore there is an urgent need to develop new targets to control the diseases Cell surface proteins which play many important roles in pathogenesis are the potential targets However those surface proteins were anchored to the cell surface by sortase a cysteine protease mainly exists in Gram-positive bacteria and mediates surface proteins forming covalent bond with bacterial cell wall via a transpeptidation reaction Thus sortases can be considered as a promising target for the development of inhibitory molecules to block the surface proteins display in cell wall In this study I have successfully determined the crystal structure of an anchored protein sortase B from C difficile and aim to use the crystal structure of sortase for future structure-based drug design against CDAD through inhibition the protease activity of SrtB Recombinant C difficile SrtB deltaN26 protein was successfully expressed in Escherichia coli and purified by Ni2+-NTA affinity chromatography C difficile SrtB deltaN26 is characterized to form monomer in solution The crystal structure of SrtB deltaN26 was solved to 2 8 ? and consisted of three ?-helices two 310-helices and eight β-strands The overall structure of C difficile SrtB deltaN26 is similar to these of B anthracis S aureus and S pyogenes However the orientations and distances between catalytic residues of Cys-His-Asp catalytic triad are different suggesting the binding-specificity of SrtB in C difficile Due to scarcity of crystal structure of sortase-substrate complex in the present literature the molecular mechanism of how sortase anchoring the surface protein to cell wall remains to be explored Therefore I have crystallized SrtB deltaN26 C209A-PPKTG complex for future structural determination Furthermore I discovered that curcumin and AAEK1 were effective inhibitors of C difficile SrtB deltaN26 by fluorescence resonance energy transfer (FRET)-based assay In summary results from my thesis studies have provided the atomic resolution of C difficile SrtB identified the effective inhibitors and crystallized SrtB deltaN26 C209A-PPKTG complex My studies have contributed to the framework toward understanding the mode of actions of sortase in Gram-positive bacteria and as a first step for the development of marketable new drug against CDAD
Date of Award2015 Jul 31
Original languageEnglish
SupervisorShu-Ying Wang (Supervisor)

Cite this

Crystal structure and functional analysis of the Clostridium difficile sortase
睿婕, 尹. (Author). 2015 Jul 31

Student thesis: Master's Thesis