Technical Reference #1755

1755.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis Katrijn Bockstael; Nick Geukens; Lieve Van Mellaert; Piet Herdewijn; Jozef Anne; Arthur Van Aerschot, Katholieke Universiteit Leuven, Microbiology, 155(1755), (2009)
Link To Paper

Abstract:
The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins making it essential for protein secretion and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1 Sip2 and Sip3) of which Sip1 lacks the catalytic lysine. In this report we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A2 were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria but also is a feasible target for biofilm-associated infections.

Materials & Methods:
Biofilm imaging by confocal laser scanning microscopy (CLSM). Aliquots (3 ml) of 1 : 50 diluted overnight culture were used to grow S. epidermidis 1457 biofilms on 35 mm glass-bottomed culture dishes coated with poly-D-lysine (MatTek). Biofilm formation was achieved as described above. At the end of the experiment medium was removed and dishes were rinsed once with 2 ml PBS. Wheatgerm agglutinin (WGA) which binds to polysaccharide intracellular adhesin (PIA) conjugated with fluorescent Alexa Fluor 633 (Molecular Probes) (Strathmann et al. 2002) was used to study matrix production in biofilms (staining red). A 160 ml volume of a 500 mg ml21 working solution of WGA in PBS was carefully applied directly on top of the biofilm. After incubation for 30 min in the dark at room temperature excess staining solution was removed by washing twice with PBS. Staining of bacterial cells within the biofilm was performed by use of the fluorescent DNA-binding stain SYTO 9 (Molecular Probes) for living bacteria (staining green) and SYTOX orange (Molecular Probes) for dead bacteria (staining yellow). Subsequent to WGA staining the biofilms were treated with 160 ml freshly prepared staining solution containing 5 mM SYTO 9 and 5 mM SYTOX orange in PBS. Biofilms were incubated for 30 min in the dark at room temperature and excess staining solution was removed by washing once with PBS. Arylomycin A2-treated and untreated biofilms were examined on a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems). Images were obtained using an HCX PL APO 663 [numerical aperture (NA) 1.20] water immersion lens and an HC PL APO CS 620 (NA 0.70) lens. Images were recorded at 476 nm excitation and 479–538 nm emission wavelengths for SYTO 9 (green channel) 561 nm excitation and 566–615 nm emission wavelengths for SYTOX orange (yellow channel) and at 633 nm excitation and 652–752 nm emission wavelengths for WGA– Alexa Fluor 633 (red channel). Digital images were recorded using the standard Leica software (Leica LAS AF version 2.0).

Microscopic Technique
confocal laser scanning microscopy

Cell Type(s)
S. epidermidis