Monolayers were washed a further three times with PBS to remove residual antibiotic and then lysed with 1 ml of ice cold sterile water. Bacterial cells were enumerated by serial dilution in PBS and plated on GM17 agar containing 5 μg/ml chloramphenicol. The remaining lysate from error prone PCR pools were inoculated into GM17 containing 5 μg/ml chloramphenicol, grown overnight, stocked at -80°C with the protocol repeated for seven passages through CT-26 cells. EGD-e

derivatives were plated onto BHI agar. Internalin A chromosomal mutagenesis in L. monocytogenes A 2 kb fragment was PCR amplified (primers IM467 and IM490) from the appropriate mutated pNZ8048binlA plasmid, with primer design incorporating the first 16 nt upstream of the inlA GTG start PF299804 mouse codon. The amplimers were digested with NcoI/PstI, ligated into complementary digested pORI280 and Selleck Ruxolitinib transformed into E. coli strain EC10B (Table 1). The plasmids pORI280 and pVE6007 we co-transformed into EGD-eΔinlA and mutagenesis preformed as described by previously [20]. The reconstruction of the inlA locus was identified by colony PCR (primers IM317 and IM318) with the integrity of the gene confirmed

by DNA sequencing. Intragastric versus intravenous infections of Balb/c mice For all murine experiments, 6-8 week old female Balb/c mice (Harlan) were used. All experiments were approved by the institutional ethics committee. Tail vein intravenous infections were conducted as described previously [18] with an inoculum comprised of equal numbers of EGD-e::pIMC3kan and EGD-e InlA m * ::pIMC3ery (2 × 104 total in 100 μl). For oral inoculation, overnight cultures were centrifuged (7,000 × g for 5 min), washed twice with PBS and resuspended at 5 × 1010 cfu/ml in PBS containing 100 mg/ml of CaCO3. A 200 μl inoculum was comprised of either a single strain (5 × 109 cfu) or a two strain mixture (5 × 109 of

each strain). Mice were intragrastrically gavaged and the progression of infection followed over a three day time course. For bioluminescent imaging, mice were SB203580 purchase anesthetized on day 1 through to day 3 with isoflurane gas and imaged in a Xenogen IVIS 100 (Xenogen) at a binning of 16 for 5 min. Mice were euthanized with spleen and livers aseptically removed, imaged (binning of 8 for 5 min) and enumerated as previously Reverse transcriptase described [18]. Results A L. monocytogenes gentamicin protection assay for murine cells Invasion into Caco-2 cells by L. monocytogenes is dependent on the expression of functional InlA [10]. We confirmed that a L. monocytogenes mutant producing InlA without the LRR and IR domain (ΔinlA) is severely compromised in invasion, while an over expressing InlA strain exhibits dramatically enhanced invasion (Figure 2). To establish an equivalent murine assay for L. monocytogenes we used monolayers of CT-26 cells (murine colonic carcinoma cell line) originally isolated from Balb/c mice chemically treated to induce tumor formation [24].