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Evaluation of the type I signal peptidase of Staphylococcus epidermidis as novel antibacterial target

Abstract number: P563

Bockstael K., Geukens N., Anné J., Herdewijn P., Van Aerschot A.

Objectives: In a time of emerging bacterial resistance there is need for novel antibacterial targets. One of the bacteria of concern is Staphylococcus epidermidis, an opportunistic pathogen associated with foreign body infections and nosocomial sepsis.

We aim to develop antimicrobial drugs against a new target, the type I signal peptidase (SPase I). SPases I have a role in protein secretion as they remove the signal peptides from secretory proteins during or after translocation across the membrane and have been shown to be essential for the viability of the cell. S. epidermidis has three type I signal peptidases, Sip1, Sip2 and Sip3.

We therefore aim to evaluate the SPase I of S. epidermidis as a novel antibacterial target and to design, synthesize and evaluate the inhibitory activity of potential SPase I inhibitors.

Methods: For all DNA manipulations standard techniques were used. SDS-PAGE was used to analyse the overproduction of the enzymes in Escherichia coli after IPTG induction. Purification of His tagged enzymes was performed under native conditions using affinity chromatography on a Ni2+-NTA agarose column. SPase expression in planktonic and sessile grown S. epidermidis was analysed by RT-PCR experiments.

Substrate analogues were rationally designed (by modelling using the structure for the E. coli signal peptidase) and synthesised by solid phase peptide synthesis using classical coupling reagents for peptide bond formation and Fmoc protected amino acids. The synthesised compounds were purified by Reversed Phase-High Performance Liquid Chromatography and analysed by High Resolution Mass Spectrometry.

Inhibitory activity against SPase I was evaluated by an in vitro inhibitor assay based on intramolecular fluorescent resonance energy transfer (FRET) using an internally quenched fluorescent peptide substrate based on a S. epidermidis preprotein.

Results: The SPase I genes of S. epidermidis were cloned, expressed and purified in E. coli and biochemically analysed. The enzymes were used to develop and optimise a FRET based in vitro assay, allowing evaluation of potential SPase I inhibitors such as rationally designed peptide substrate analogues.

Conclusion: Due to their unique properties, SPases I seem to be an attractive target for the development of new antimicrobial drugs. A FRET based in vitro assay was developed and used for the evaluation of potential SPase I inhibitors.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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