This DNA fragment was cloned as a BamHI/NdeI restriction selleck chemical fragment in the expression vector pET3a (Novagen) and the clones were verified by DNA sequencing. Escherichia coli BL21(DE3)pLys was transformed with the resulting construct. Following isopropyl β-d-1-thiogalactopyranoside (IPTG) induction (100 μM IPTG) of the BL21 strain containing the Lcl overexpression plasmid, the recombinant protein was purified on a Ni2+-NTA agarose column under denaturing conditions (8 M urea). Following sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE), the protein was electroeluted from the gel and with this purified protein antibodies (AB) were raised against L. pneumophila Lcl in pfd:Hollander rabbits. The specificity of the antibodies was tested using a total cell lysate of L. pneumophila. The antibodies were used to detect Lcl after SDS-PAGE using Western blot analysis with anti-rabbit antibodies as secondary antibodies and NBT/BCIP (Roche) for signal detection. The denatured proteins were refolded through dialysis Selleckchem Lapatinib and the concentration was determined

using a bovine serum albumin (BSA)-standard curve. The lpg 2644 gene, containing 19 repeat units of 45 nucleotides, was amplified from the L. pneumophila ATCC33152 genomic DNA with the primer pair 5′-TACATATGATACATCGAAATAAAGTCC-3′ and 5′-TAGAATTCTTAAAAGGCTCTTACAGC-3′. This fragment was cloned as an NdeI/EcoRI restriction fragment in the shuttle vector pMMBN and electroporated in L. pneumophila Philadelphia-1 [wild type (WT)/pMMBNlcl]. Expression of the lcl gene was induced by addition of 100 μM IPTG. Cloning procedures led to a spontaneous recombination of the VNTR region, resulting in an lcl gene containing 14 repeat units designated WT/pMMBNlcl(14). Fractionation of L. pneumophila Philadelphia-1 cultures was performed as described before (Vranckx et al., 2007). Briefly, the supernatant was concentrated by trichloroacetic acid

(TCA) precipitation (20% TCA final concentration) and the cells were lysed in a French pressure cell. To extract the inner membrane proteins from the membranes, the sediment was resuspended in 1.5 mL 10 mM Tris (pH 7.5) containing 1.5% sarkosyl and centrifuged. The outer membrane proteins were Galeterone resuspended in 500 μL 10 mM Tris, pH 7.5, and 10 mM EDTA, containing 1% Triton X-100. The quality of the cellular fractions was controlled by testing for the presence of DnaK, a cytoplasmic protein, LepB, an inner membrane protein, and Lpa, an outer membrane protein. WT bacteria grown to the stationary phase were added to a monolayer of A549, macrophage-like cells or A. castellanii (5 × 105 cells per well) at a multiplicity of infection (MOI) of 100. Bacteria overexpressing Lcl were added to a monolayer of A549 or macrophage-like cells also at an MOI of 100. For sampling, after 30 and 60 min, the supernatant was removed and the wells were washed three times with medium to remove the extracellular bacteria.