Nesse et al. Acta Veterinaria Scandinavica 2011, 53:44 http://www.actavetscand.com/content/53/1/44 Table 1 Primers used for RT-PCR Gene Acc. no/locus tag rck CP001362.1 /STM14_5534 srgE AE006468.1/STM1554 Primers 5’ - 3’ F: forward R: reverse F: GTTGTATCCCGGCATGCTGAT R: ATATGCCCAGAGCCGGATAGAG F: GTAATGTCAATTGCGGCATGG R: CGGAGCAGTTGGTCAAGGATT initial denaturation at 95°C, thereafter 26, 28 and 31 cycles of 95°C 40s, 60°C 30s and 72°C 40s, followed by 7 min final extension at 72°C. The primers used are listed in Table 1. The PCR fragments were separated by gel electrophoresis and examined both visually and using BioNumerics® software (Applied MathsBVBA, Belgium). The best measurements of quantitative differences were obeserved at 28 cycles. Results and Discussion Both the S. Typhimurium wildtype strain ATCC 14028 and its isogenic sdiA mutant displayed invasion of the epithelial cell line HEp-2 at 37°C without addition of AHLs (0.5 - 2.5 * 105 cfu per well, depending on the day to day variation). However, when 1 μM of C8-AHL or C6-AHL was added, the S. Typhimurium wildtype strain displayed statistically significant higher cell inva- sion (approximately two fold) with both AHLs, as com- pared to the invasion without AHL (Figure 1). No such log10 fold increase 0,40 Page 3 of 5 differences were observed when testing the isogenic sdiA mutant strain, indicating that the increased cell invasion responses were sdiA dependent. To date, SdiA is known to activate two loci, contain- ing a total of seven genes [5,8]. One locus, the rck operon, is located on the virulence plasmid and contains six genes. The second locus is located on the chromo- some and encodes a single gene, named srgE. Several different AHLs, including C6-AHL at the concentration of 1 μM, have earlier been shown to activate promotors of the genes rck and srgE in an sdiA-dependent manner in Salmonella [5,8]. To see if these genes were activated in our experiment, we studied the expression of the genes under similar experimental conditions as the cell invasion experiments using a semi-quantitative PCR (Figure 2). For both genes, we observed a low level of sdiA- and AHL-independent expression, as earlier reported for rck [5,8]. In addition, the wildtype S. Typhi- murium strain displayed an increased expression of both rck and srgE when 1 μM of C8-AHL or C6-AHL was added, whereas its isogenic sdiA mutant did not. The results indicate that SdiA acted as an AHL induced transcriptional regulator under our experimental conditions. Consequently, the results from the present study show that a S. Typhimurium wildtype strain did respond to both C8-AHL and C6-AHL with increased epithelial cell C6-AHL C8-AHL 0,30 0,20 0,10 0,00 -0,10 S . Typhimurium 14028 -0,20 Figure 1 The effect of N-hexanoyl-DL-homoserine lactone (C6-AHL) and N-octanoyl-DL-homoserine lactone (C8-AHL) on cell invasion by Salmonella expressed as mean fold increase (log10 transformed). Bars show 95% confidence interval. When the value 0 is not included in the confidence interval, the increase is considered statistically significant, i.e. p <0.05. S . Typhimurium 14028 sdiA mutant