Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 Page 3 of 13 Figure 1 Relative number of intracellular bacteria in each living U937 cell. The cultured HLA-B27 or HLA-A2-transfected U937 cells were infected with complement-opsonized S. Enteritidis PT4 KS8822/ 88 and the incubation was continued at 37°C for 1, 4, 8 or 24 h after infection. At the indicated time points, the host cells were lysed and the number of bacteria per cell was counted and reported as colony forming units (CFU). For both cell lines, the number of intracellular bacteria at 1 h after infection was designated 1. The numbers of intracellular bacteria at the following indicated time points are shown as fold changes relative to the number at 1 h after infection. Values are mean ± standard deviation of duplicate samples from a representative experiment out of three with similar results. * P < 0.05 versus S. Enteritidis-infected HLA-A2 transfectants. Data were compared using Student’s paired 2-tailed t-test. expression at 2 h post infection when compared to the transcriptome in LB broth culture. At 8 h post infection, the expression of 1,352 genes of Salmonella grown in HLA-B27 cells and of 1,559 genes of Salmonella grown in HLA-A2 cells showed statistically significant differ- ences from Salmonella grown in LB broth. The genes that showed significant expression differences and were up- or down-regulated more than two-fold for each individual comparison are listed in Additional file 1: Supplemental Tables 1-6. The categories of Salmonella genes showing differential expression between HLA-B27 cells and LB and between HLA-A2 cells and LB at 2 h post infection can be found in Figure. 2. In particular, the functional categories analysis showed large differ- ences in the expression of genes required for cell moti- lity and secretion, flagellae and chemotaxis and oxidative phosphorylation. These differences in gene expression reflect bacterial adaptation to the intracellu- lar environments. To confirm the microarray data, quantitative real time RT-PCR was performed on six genes selected from dif- ferent functional categories and showing differential up- Figure 2 Differential expression of S. Enteritidis genes divided into functional groups at 2 h time point. Bars indicate percentages of genes in each group that showed significant changes in expression in HLA-A2 cells (A) and HLA-B27 cells (B), which were compared to cells grown in LB. The white bars indicate the proportion of up-regulated genes and the black bars the percentage of down-regulated genes for each group. or down-regulated expression between HLA-B27 and A2 cells: mgtC, rpoE and ssaG (up-regulated); fliC, fumC and nuoA (down-regulated) (Figure. 3). RT-PCR was performed using the same bacterial RNA from HLA- B27 cells, A2 cells and LB broth, as was used in micro- array experiments. It is important that the genes selected covered the whole scale of the microarray data from high expression level to medium and to low expression level, as well as in differential expression from high fold changes to low fold changes in U937 cells vs. in LB, among up-regulated and down-regulated genes, respectively. The quantity of cDNA for each gene was obtained after normalization to the levels of rfaH cDNA, which was chosen as a control since rfaH is expressed stably at moderate levels under most of the conditions tested (see Additional file 2: Supplemental Table S8). The RT-PCR results of the six genes studied were consistent with the microarray data (Figure. 3), but due to the small number of genes studied with RT-PCR, data may be considered preliminary.