monocytogenes InlA over-expressing strain and ΔinlA strain were c

monocytogenes InlA over-expressing strain and ΔinlA strain were compared (Figure 2) and was also seen in experiments in the L. lactis background (Figure 3). These

results could be due to the high level of inlA expression from the Pnis and Phelp promoters, amplifying the differences in InlA on the surface of L. lactis and L. monocytogenes cells (Figure 2 and 3). We interpret these results as evidence of a specific interaction between InlA and a cell surface receptor on CT-26 cells which stimulates bacterial cell entry. To summarise, we have established a gentamicin protection assay, capable of discriminating InlA mediated invasion into selleck compound a murine cell line. Generation and screening of a random bank of InlA LRR mutants To generate diversity within the inlA gene we applied error prone PCR to the LRR region (between naturally occurring BglII/BstXI sites – Figure 1a). Four separate banks were created containing different levels of mutation frequency, each containing about 40,000 L. lactis clones. Initial assessment by DNA sequencing of ten clones from each

bank identified ICG-001 research buy mutations throughout the LRR region with the level of mutation correlating with the concentration of input template DNA for the error prone PCR (data not shown). To identify positive mutations, pools were invaded through CT-26 cells en masse as detailed in Figure 4. Sequential passages through CT-26 cells were required to remove the background functional InlA Fenbendazole from the pools (Figure 5). Of the four banks only the highest mutation frequency resulted in an initial recovery below that of wild type InlA, which suggested that a significant number of clones contained inactivating mutations. From passage two through six a significant enrichment in positive mutations was observed, with a leveling off at passage seven (Figure 5).

From passage six, eight clones from each bank were sequenced (Table 2) and assayed individually using both CT-26 and Caco-2 cells (Figure 6). All clones exhibited enhanced entry into CT-26 cells while no apparent differences for cell entry into Caco-2 cells were observed (compared to L. lactis InlAWT). However, no clones were identified which were capable of matching the level of L. lactis InlA m * mediated entry into the murine cells. Sequence analysis revealed that 23 of the 32 clones contained amino acid changes in residues involved in direct interaction with CDH1. Of the four banks, only the lowest mutation frequency contained multiple clones with the same mutation (Gln190Leu), with this single amino acid change also found in one clone from an additional bank (Table 2). Figure 4 Enrichment protocol for the selection of mutations in InlA conferring enhanced invasion of L. lactis into CT-26 cells. Cultures of L. lactis + pNZB containing (i) inlA WT (ii) inlA m * or (iii-vi) 4 banks of clones with different levels of mutation in the LRR of inlA WT were induced with nisin and assayed for invasion into CT-26 cells by gentamicin protection assay.

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