Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 SP or SCV favours the survival and replication of Sal- monella in macrophages. Even so, Salmonella encounter intracellular host defence mechanisms, including reac- tive oxygen and nitrogen species (ROS and RNS), anti- microbial peptides, lysosomal enzymes, and adaptive immune responses [10,12]. In order to survive in the host and to avoid clearance by the host immune system, Salmonella express virulence factors to deal with this stressful environment [14]. Many virulence genes of pathogenic bacteria are located in large multigene chromosome regions termed pathogenicity islands (PAIs) [15]. In Salmonella, they are called Salmonella pathogenicity islands (SPIs) [16]. Two SPIs, SPI-1 and SPI-2, encode structurally similar but functionally distinct type III secretion systems (T3SS), specialized protein export machineries that Sal- monella uses to deliver virulence proteins into the cyto- sol of host cells [17]. The SPI-1-encoded T3SS is active extracellularly. SPI-1 mediates invasion into non-phago- cytic cells [18], and it is required for the intestinal inflammatory responses [19]. The SPI-2 virulence genes are expressed intracellularly and are required for the survival of bacteria in macrophages and systemic infec- tions. SPI-2 mutant strains are dramatically attenuated, showing a 104-fold reduction in virulence in LD50 in the murine salmonellosis model [16] and impaired intracel- lular replication and survival in macrophages [20,21]. HLA-B27 confers a very strong genetic predisposition towards the development of a group of rheumatic disor- ders called spondyloarthropathies (SpA), including anky- losing spondylitis (AS) and ReA. HLA-B27-positive individuals have a five-fold higher incidence of ReA than the general population [22,23]. ReA occurs follow- ing certain infections, e.g. those caused by Salmonella and Yersinia pathogens [7,8,24,25]. Expression of HLA- B27 also increases the risk that the patient will suffer a more severe and prolonged disorder [6,8]. The interac- tion between ReA-triggering bacteria and HLA- B27-positive subjects is abnormal and leads to increased persistence of the causative microbes/microbial antigens in HLA-B27-positive patients [26-28]. The interaction between HLA-B27 molecules and arthritogenic microbes was investigated more thoroughly using in vitro infected cells. Experiments investigating the inva- sion of HLA-B27 cells by Gram-negative bacteria, including Salmonella, are inconclusive. Studies have shown either decreased [29] or similar [30] levels of invasion of HLA-B27-transfected murine L fibroblasts compared with control L cell lines (L cells transfected with other MHC class I genes) or increased invasion of HLA-B27-transfected intestinal epithelial Henle-407 cells by Salmonella [31]. Once inside the host cells, Salmonella is able to replicate more quickly [32,33], and is eliminated more slowly, in HLA-B27-positive cells Page 2 of 13 [30,34] than transfected control cells. The survival and persistence of Salmonella in the intracellular environ- ment is associated with bacterial gene expression [35]. However, little is known about Salmonella gene expres- sion in association with HLA-B27 during bacterial infec- tion and persistence. Results and Discussion Cell surface expression of HLA-B27 and HLA-A2 molecules The transfected HLA-B27 and HLA-A2 were always expressed on the surface of the respective cells, as detected by immunofluorescence in new batches of the cell lines (data not shown). The level of expression of the transfected molecules in U937 cells was similar to that of HLA-B51, one of the MHC class I molecules endogenously expressed by U937 cells [34]. In addi- tion, the surface expression levels of the transfected molecules corresponded to the levels of those mole- cules endogenously expressed on peripheral blood monocytes [34]. Increased replication of Salmonella in HLA-B27-positive U937 cells We used the in vitro model of infection established ear- lier in our laboratory to monitor growth of S. Enteritidis in macrophage-like U937 cells [32-34]. Cells transfected with HLA-B27 or HLA-A2 were infected with comple- ment-opsonized salmonellae. HLA-A2 transfected cells were used as a negative control since it is a common tis- sue antigen that is not related to the development of ReA [34]. At 1, 4, 8, and 24 h post infection, the host cells were lysed and the number of living intracellular bacteria per cell was determined by counting the num- ber of colony forming units (CFU) (Figure. 1). Consis- tent with our previous results, no difference in the uptake of Salmonella was observed between the two cell lines (1 h post infection) [34]. However, more bacteria were recovered from HLA-B27-expressing cells than HLA-A2-expressing cells at 8 h and even up to 24 h post infection, suggesting that HLA-B27-transfected cells become permissive for intracellular Salmonella sur- vival and replication, which is also consistent with our previous studies [32-34]. Global gene expression profiles of S. Enteritidis in HLA- B27- and HLA-A2-transfected U937 cells The ‘SALSA’ genomic Salmonella serovar microarray containing 5080 Salmonella oligonucleotides was used to monitor gene expression in Salmonella grown in the host cells [36]. Gene expression was compared between Salmonella grown in U937 cells and in LB broth. In total, 1,388 genes of Salmonella grown in HLA-B27 cells and 1,049 genes of Salmonella grown in HLA-A2 cells showed statistically significant differences in