Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 intracellular bacterial growth environment during the infection of macrophages. Expression of SPI-2 genes, which play a key role in the increased persistence of Salmonella in macrophages, was up-regulated in HLA- B27 cells compared to HLA-A2 cells. DNA replication, energy production and nutrient metabolism were also increased, suggesting that more bacterial physiological activities occurred in HLA-B27 cells. This is consistent with the increased replication and survival of Salmonella in HLA-B27-transfected macrophages. Interestingly, expression of many SPI-2 genes, energy production and nutrient metabolism are also increased in epithelial cells where Salmonella replicates intracellularly [64], whereas e.g. Cyo and Cyd terminal oxidases, which use oxygen exclusively as terminal acceptor, are strongly downregu- lated in Salmonella inhabiting restrictive cells, fibro- blasts [65] and HLA-A2 cells (Additional file 1: Supplemental Table S2-2 and S4-2). Mutagenesis of the SPI-2 genes demonstrated the cru- cial role of SPI-2 in the survival and replication of Salmonella in HLA-B27 cells, as has been seen in other macrophages [18,19]. The intracellular replication and survival of the strains bearing deletion of either ssaR or sspH2 was impaired in comparison with wild-type PT4 (Figure. 5A). Both proteins participate in the mainte- nance of the SCV membrane, which is important for the survival of Salmonella in infected macrophages. Increased growth of the ssaN mutant in HLA-B27-posi- tive cells might indicate a special connection between this mutant and HLA-B27-positive cells, and may even be relevant to the pathogenesis of HLA-B27-associated ReA. HLA-B27 modifies the host cell’s signalling pathways during Salmonella infection. Our group recently demon- strated that mitogen activated protein kinase (MAPK) p38 is involved in Salmonella replication in U937 cells, and that the p38 MAPK pathway is deregulated in HLA-B27-transfected cells [33]. Salmonella uses type III secretion systems (T3SS) encoded in SPI-1 or -2 to deli- ver virulence proteins into host cells, which subse- quently interfere with the host cell’s signalling pathways [15,66,67]. Further experiments will be required to determine how Salmonella genes, particularly SPI-2 genes, affect intracellular signalling pathways and cause increased replication of bacteria in HLA-B27 cells. Nevertheless, these findings give new insights into the disturbed microbe-host interaction in HLA-B27-positive cells. Methods Cell lines and transfections The human monocytic U937 cell line was obtained from ATCC (Rockville, USA). The full-length 6-kb genomic clone of human HLA-B*2705 DNA [68] in the vector Page 8 of 13 pUC19 and the full-length 5.1-kb genomic clone of human HLA-A2 DNA [69] in the vector pUC9 were kind gifts from Dr. Joel D. Taurog. For transfection, U937 cells were suspended in RPMI 1640 containing 1.8 mM L-glutamine and 1 mM sodium pyruvate (Gibco, Paisley, Scotland), and were then cotransfected by elec- troporation with the vectors carrying either HLA- B*2705 or HLA-A2 DNA with the plasmid pSV2neo (to provide resistance to geneticin [G-418]) (CalBio- chem, Darmstadt, Germany). Stable transfectants were selected with 0.5 mg/ml geneticin and the expression of transfected HLA-B27 or -A2 molecules on the surface of U937 cells was examined by immunofluorescence (IF) and flow cytometry as described previously [34]. The transfected cell lines were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS; PAA laboratories, Linz, Austria), 1.8 mM L-gluta- mine and 50 μg/ml gentamicin (Gm) (both from Biolo- gical Institutes, Kibbutz Beit Herennek, Israel) at 37°C in a humidified atmosphere of 5% CO2/95% air. Cell cultures were tested to confirm freedom from myco- plasma contamination. Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids used in this study are listed in Table 2. The wild type S. Enteritidis strain was originally isolated from the stool of a patient with Salmonella-triggered ReA [34], and was typed as phage type 4 (PT4) and named S. Enteritidis PT4 KS8822/88. The S. Enteritidis and Escherichia coli strains were routinely grown in Luria-Bertani (LB) broth at 37°C. Bacteria carrying a temperature-sensitive plasmid (pKO- BEGA) were grown at 30°C. Media were supplemented with 100 μg/ml ampicillin (Amp) (Sigma) and 30 μg/ml kanamycin (Km) (Sigma) as required. Cell infection model For infection, the U937 cells were diluted to 1.0 × 106 cells/ml and then seeded in tissue culture flasks (75 cm2) or 24-well plates (Greiner, Germany). Prior to bacterial infection, the cells were cultured with phorbol myristate acetate (PMA; Sigma) for 24 h to differentiate them toward more mature macrophage-like cells. Two hours before infection, the adherent cells were washed with Hank’s balanced salt solution (HBSS) and then overlaid with prewarmed RPMI 1640 supplemented with 10% human AB serum (Finnish Red Cross, Helsinki, Finland). The cells were then co-cultured with Salmo- nella at a multiplicity of infection (MOI) around 50:1. After 2 h of infection, the cells were washed three times with HBSS to remove non-adherent bacteria and over- laid with fresh RPMI 1640 containing 50 μg/ml Gm to kill extracellular bacteria. The Salmonella-infected cells were then incubated at 37°C for 1, 4, 8 or 24 hours as