Our characterization of the FPI mutant ΔpdpC demonstrates that is exhibits a unique phenotype compared to other FPI mutants since it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, but still efficiently triggered secretion of IL-1β and markedly induced LDH release. The findings implicate that a
AZD6244 manufacturer thorough understanding of the function of PdpC will provide important understanding behind the unique intracellular life cycle of F. tularensis. Methods Bacterial strains, plasmids, and growth conditions Bacterial strains and plasmids used are listed in Additional file 1: Table S2. Escherichia coli strains were grown either in Luria Bertani broth (LB) or on Luria agar plates (LA) at 37°C. F. tularensis was cultured either in Chamberlain’s medium [46] or in TSB at 37°C, 200 rpm, or on modified GC-agar at 37°C, 5% CO2. When required, kanamycin (50 μg/ml for E. coli or 10 μg/ml for F. tularensis), carbenicillin (100 μg/ml), tetracycline (10 μg/ml), polymyxin B (50 μg/ml) or chloramphenicol (25 μg/ml for E. coli, 2.5 μg/ml for F. tularensis) was added to the medium. The ΔiglA or ΔiglC mutants were used as controls for phagosomally located bacteria. Both have previously been characterized in detail by us and others, and their phenotypes are indistinguishable in
that they are avirulent and show no phagosomal escape or intramacrophage replication [16, 47–49]. Bioinformatic studies The bioinformatic analysis was performed using the following Selleckchem Tucidinostat web-based tools: PSORTb (http://www.psort.org/psortb/index.html) for prediction of localization, TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) to find putative transmembrane regions, SMART (http://smart.embl-heidelberg.de) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for identifying conserved domains, and CBS prediction servers (http://www.cbs.dtu.dk/services) to find
a lipoprotein signal, signal peptides or secretion signals. Construction of expression vectors and the bacterial-two-hybrid (B2H) assay For the bacterial two-hybrid assay, PCR-amplified Tangeritin iglE, iglF, iglG, iglH, iglI, iglJ, pdpC, pdpE, iglD, pdpA, pdpD, fevR, and pmrA were initially cloned into the pCR4-TOPO TA cloning vector to facilitate sequencing, and subsequently introduced as NdeI/NotI fragments into the IPTG-inducible plasmids pACTR-AP-Zif and pBRGPω [50]. For alleles containing intrinsic NdeI sites (iglJ, fevR, pmrA), these were mutated by overlap PCR prior to cloning. Since PdpD is significantly truncated by an in-frame stop codon in LVS, we used F. tularensis subsp. novicida U112 as template in the overlap PCR reaction to amplify full-length pdpD without its intrinsic NdeI site. Primer combinations used to construct the B2H alleles are listed in Additional file 1: Table S3. Plasmids were transferred into E. coli DH5αF’IQ (Invitrogen AB, Stockholm, Sweden) by Selleck MK-8931 electroporation.