Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 Page 6 of 13 Figure 5 Intracellular survival and replication of SPI-2 mutant Salmonella strains in U937 cells. The cultured HLA-B27 or HLA- A2-transfected U937 cells were infected with complement- opsonized S. Enteritidis PT4 KS8822/88 wild type or mutants and incubation of the bacteria-infected cells was continued at 37°C for 1, 4, 8 or 24 h after infection. At the defined time points, the host cells were lysed and the number of bacteria per cell was reported as colony forming units (CFU). The numbers of bacteria in each HLA-B27 cell (A) or HLA-A2 cell (B) at the indicated time points are shown. Values are mean ± standard deviation from three independent experiments with duplicate samples. * P < 0.05, statistically significant differences between the wild type and mutant strains. Data were compared using Student’s paired 2-tailed t-test. formation of Sifs by ssaR mutants may account for the attenuation of this strain. The effector protein SspH2 is co-localized with vacuole-associated actin polymeriza- tion involved in the maintenance of SCV membrane integrity [46]. The sspH2 mutant was also attenuated more than 4-fold in HLA-B27 cells at both 8 and 24 hours after infection (Figure. 5A). Unexpectedly, the mutant carrying the ssaN deletion showed dramatically increased proliferation in HLA-B27 cells: 1.4-fold at the 8 h and 3.9-fold at the 24 h time points (Figure. 5A). The increased growth of the ssaN mutant in HLA-B27 cells might indicate that this mutant has good fitness in HLA-B27-positive U937 cells. The previous study by Eriksson et al [47] showed that the selection for S. Typhimurium mutants performed in murine macro- phage-like J774-A.1 cells provided bacterial variants cap- able of a selective downregulation of nitric oxide (NO) expression. These variants showed increased host fitness. This is especially interesting, as in our model with mur- ine L cells [30], iNOS activity and NO production were decreased in HLA-B27 positive cells, which correlated with increased S. Enteritidis growth. However, human U937 cells show very low iNOS activity and produce only small amounts of NO in general, and no difference between HLA-B27-positive and control cells was detected in this respect [48]. So the increased prolifera- tion of the ssaN mutant in HLA-B27 cells, and no effect on growth in HLA-A2 cells (Figure 5B), perhaps indicates another specific and delicate relationship between ssaN and HLA-B27. These observations demonstrated a delicate balance between bacterial growth and pathogenesis. Regulation of bacterial DNA synthesis by HLA-B27 As Salmonella replicates more in HLA-B27-transfected cells than in HLA-A2 transfectants, it is not surprising that genes involved in DNA synthesis are up-regulated in HLA-B27 cells compared to HLA-A2 cells. DNA polymerase III (pol III) holoenzyme is a major replicase responsible for DNA synthesis [49]. It is a multiprotein complex containing over 10 distinct subunits. The a subunit encoded by dnaE is one component of the cata- lytic core (aεθ) of the pol III holoenzyme and mutations affecting the a unit result in reduced growth of S. enter- itica serovar Typhimurium [50]. The expression of the gene dnaE was induced 2.7-fold in HLA-B27 cells com- pared to HLA-A2 cells at 8 h time point (Table 1). Other dna genes including dnaA encoding DNA replica- tion initial protein, dnaB encoding putative replicative DNA helicases, and dnaN encoding b-subunit of DNA polymerase III were up-regulated 1.9-, 1.4-, and 2.0-fold, respectively. However, dnaQ encoding ε-subunit of DNA polymerase III was expressed at the similar level in two cell lines (see Additional file 1: Supplemental Table S1). The up-regulation of most dna genes in HLA-B27 cells compared to HLA-A2 cells might indi- cate more DNA replication in HLA-B27 cells. Two genes (gyrA and gyrB) required for the production of the heterotetramer-DNA gyrase were also up-regulated in HLA-B27 cells compared with the control cells. DNA gyrase is an essential enzyme for bacterial DNA