Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 Page 4 of 13 Figure 3 Validation of microarray results by quantitative real time RT-PCR. The fold changes in gene expression are shown when S. Enteritidis growth in HLA-A2 or HLA-B27 cells was compared to growth in LB broth. The expression levels of selected up-regulated genes (A) or down-regulated genes (B) are shown in logarithmic scale. The experiments were repeated at least three times with similar results. Values are mean numbers ± standard deviations generated from three independent experiments. Correlation coefficients (R) for comparisons of two techniques are: up-regulated genes; 0.977 (A2/LB) and 0.959 (B27/LB), down- regulated genes 0.530 (A2/LB) and 0.620 (B27/LB) (see Additional file 2: Supplemental Table S7). Salmonella gene expression is regulated by HLA-B27 at the 8 h time point To determine whether bacterial gene expression plays a role in Salmonella replication/persistence in HLA-B27 cells, we assessed the transcriptome of S. Enteritidis in the two cell lines. Differential expression of the genes betweeen HLA-B27 cells and HLA-A2 cells is based on the comparison of the gene expression in HLA-B27 cells to LB and the gene expression in HLA-A2 cells to LB. For example, for the gene whose expression is 6-fold in HLA-B27 cells compared to LB, and 2-fold in HLA-A2 cells compared to LB, the expression is 3-fold in HLA- B27 cells compared to HLA-A2 cells. No genes showed significant differences when the transcriptomes of Salmo- nella grown in HLA-B27- and HLA-A2-transfected cell lines were compared at 2 h post infection. This is consis- tent with the similarity observed in the intracellular growth of Salmonella at this time point. In contrast, 118 genes showed significant differences in expression between Salmonella grown in HLA-B27 cells and in HLA-A2 cells at 8 h post infection (86 genes were up- regulated and 32 genes down-regulated (see Additional file 1: Supplemental Tables S6-1 and -2, Figure 4 and Table 1). Bacterial gene expression reflects the environ- mental conditions in which the bacteria reside. The difference between bacteria grown in HLA-B27 and HLA-A2 cells suggests that HLA-B27 modulates the intracellular environment and causes changes in Salmo- nella gene expression during macrophage infection, resulting in increased survival and replication in HLA- B27 cells. This modulation may be due to changes in host cell signalling pathways [33,37]. This is the first observation showing that Salmonella gene expression is influenced by HLA-B27. The analysis of functional cate- gories revealed that the groups of genes most altered in the presence of HLA-B27 included those involved in Sal- monella virulence, DNA replication, energy conversion and metabolism, and uptake and metabolism of nutrients. Up-regulation of SPI-2 genes may prevent damage by ROS SPI-2 genes have been shown to make Salmonella cap- able of growing intracellularly by avoiding killing by reactive oxygen species (ROS) [38]. During Salmonella replication in macrophages, the innate defence systems of the host cells are triggered to limit the infection. ROS produced by phagocyte NADPH oxidase (phox) are the most effective anti-bacterial agents [39]. NADPH oxidase catalyzes the univalent reduction of molecular oxygen and produces superoxide, which has modest Figure 4 Differential expression of S. Enteritidis genes between HLA-B27 and HLA-A2 cells divided into functional groups at 8 h time point. Bars indicate percentages of genes in each group that showed significant changes in S. Enteritidis gene expression in HLA-B27 cells compared to in HLA-A2 cells. The white bars indicate the proportion of up-regulated genes and the black bars the percentage of down-regulated genes for each group.