The DNA fragment was cut with SmaI and cloned into the vector pUC18, leading to the plasmid pUC18-spa. The fragment was then cut and cloned into the plasmid pUC18-Phly using the NsiI and XmaI restriction sites. The fragment Phly-spa was PCR amplified by the Primers M13 universe 2 (5′-GTAAAACGACGGCCATGGC-3′) and M13 rev (5′-CAGGAAACAGCTATGAC-3′) to introduce a NcoI restriction site. The fragment was then cloned into plasmid pLSV101-intAB [31] using the restriction sites NcoI and SacI. The resulting plasmid pLSV101-intAB::Phly-spa was transformed into L. monocytogenes ΔtrpS,inlA/B × pFlo-trpS [32] and L. monocytogenes ΔtrpS,aroA,inlA/B × pFlo-trpS [aroA attenuated
as described www.selleckchem.com/products/BEZ235.html in 33] and a homologous recombination technique was used to construct a deletion mutant [34]. Because trpS bearing plasmids are fully stable in the ΔtrpS mutant without the addition of antibiotics this strain was used for mutant generation. Western blot analysis L. monocytogenes protein extracts were prepared as described [35]. Surface proteins were extracted by incubation in 1% sodium dodecyl sulfate (SDS) for 20 min. Blotted proteins were probed with a polyclonal
goat antibody against Protein A (Biomeda, CA, USA) or polyclonal rabbit antibody against murine serum albumin (ab19196 – abcam, UK). Secondary Peroxidase-conjugated antibodies and ECL Western blot detection reagent (Amersham Biosciences, Germany) were used for visualization of bands. Analysis of bacterial protein A surface expression Bacteria were washed in PBS and incubated for 1 h at 25°C with polyclonal FITC-conjugated rabbit-anti-goat immunoglobulin G (H+L, Sigma, LOXO-101 order Germany) for
flow cytometry or polyclonal rabbit antibody directed against ovalbumin (C6534, Sigma, Germany) for immunofluorescence microscopy. Controls were incubated with PBS. Bacteria were washed 2-3 times with PBS and analyzed using an Epics XL flow cytometer (Beckman Coulter) or further incubated with FITC-conjugated OVA (Molecular Probes, Germany). After repeated washing, bacteria were loaded on microscope slides and analyzed by fluorescence microscopy (Leica, Germany). Antibody-coating, crosslinking and serum treatment of L. monocytogenes For antibody-coating, 5 × 108 CFU were washed with PBS (pH 8.2) and resuspended in 100 μl PBS containing Adenosine triphosphate 2.5 μg of Cetuximab (Merck, Germany) or 2.37 μg of Trastuzumab (Roche, Germany), respectively. Alexa Fluor labeled antibodies were generated using the Apex Antibody labeling kit (Invitrogen) following the manufacturers guidelines. The bacteria were incubated under vigorous shaking for 45 min at room temperature (RT). Bacteria were washed with PBS (pH 8.2) and diluted for further use. Crosslinking of antibodies to SPA on the surface of Lm-spa+ was performed using dimethyl pimelinediimidate dihydrochloride (DMP, Akt inhibitor Biochemika Fluka, Germany). Freshly prepared DMP in PBS (pH 8.2) was added at a final concentration of 0.65 mg/ml to the antibody coating reaction.