lactis were included for comparison. The graph is of the data from one experiment. Characterization of murinized L. monocytogenes: competitive index assays Four inlA sequences conferring enhanced invasion into CT-26 cells

were selected to be re-created in the chromosome of L. monocytogenes EGD-e. The mutations constituted Idasanutlin manufacturer two single aa changes for EGD-e A (Asn259Tyr) and EGD-e B (Gln190Leu). While three aa changes were introduced into EGD-e C (T164A, K301I, Q303E) and EGD-e D (S173I, L185F, L188I). These mutations were chosen based on the frequency of isolation in L. lactis (EGD-e B and C), the ability to attribute the phenotype to an aa change (EGD-e A) and the isolation of mutations all confined within one LRR (EGD-e D). A fifth strain was also created based on the Lmo-InlAm mutation [18], except with

Listeria optimized codons for 192Asn and 369Ser, and was used as a positive control (EGD-e InlA m *). Sequencing LY2228820 datasheet confirmed the integrity of the newly introduced mutations, with equivalent levels of InlA expressed on the surface of the strains as compared PXD101 research buy to EGD-e (assessed by western blot – data not shown). InlAm strain (termed EGD-e InlA m *) was compared to the parental EGD-e strain for invasion into Caco-2 and CT-26 monolayers. No differences in invasion (Figure 7a) or adherence (data not shown) were observed to Caco-2 cells, while the invasion of EGD-e InlA m Resveratrol * was significantly higher than EGD-e into CT-26 cells. We then compared the virulence of EGD-e and EGD-e InlA m * by competitive index (CI) assays via the intravenous (i.v.) (Figure 7b) or intragastric (i.g.) (Figure 7c) route in Balb/c mice. For i.v. inoculated mice, no differences in the kinetics of infection were observed for either strain (Figure 7b). This confirms that the two amino acid changes in InlAm do not impact on the virulence of EGD-e InlA m * once

the gastrointestinal tract is bypassed. However, EGD-e InlA m * was significantly more virulent when infected by the i.g. route, with higher counts obtained from livers and spleens and a significantly higher CI value (p < 0.001) for both day two (Liver 28.9, Spleen 10.6) and day three (Liver 24.9, Spleen 7.7 – Figure 7c). Neither strain was recovered form the liver nor spleen at day one post infection. Subsequent competitive index experiments were conducted by the i.g. route comparing EGD-e InlA m * against the strains expressing the InlA mutations identified by the CT-26 cell screen (Figure 7d). Of the four recreated strains, only EGD-e A (N259Y) gave a higher CI than EGD-e in the liver (0.19 vs 0.05) whereas identical values (0.12) were obtained for the spleens. Further experimentation will be required to access the contribution of the N259Y mutation, and it would be intriguing to see if the recombination of this mutation into EGD-e InlA m * would further enhance murine pathogenicity.