Investigating the role of tetraspanin proteins in Salmonella infection

Tetraspanin proteins are a family of transmembrane proteins that are broadly expressed on different tissues and in multi-cellular organisms. Their main characteristic is the ability to organize multi-molecular complexes through their association with different proteins and lipids to form tetraspanin enriched microdomains or TEMs. These microdomains have important roles in many cell functions, cancer malignancy and infectious diseases. However relatively few studies have investigated tetraspanin proteins in bacterial infection. Recently, tetraspanins have been shown to be involved in bacterial adhesion and invasion of human epithelial cell lines and human primary monocyte derived macrophages (MDMs). Specific monoclonal antibodies (mAb) and soluble recombinant versions of the large extracellular domains (EC2s) of tetraspanins were shown to inhibit adhesion of various bacterial species to human cells, possibly by disrupting TEM (Green et al., 2011). Therefore, treatments that target tetraspanins could offer a potential therapeutic alternative to antibiotics, in infections where resistance is an increasing problem. In this study, we further investigate the role of tetraspanin proteins in Salmonella enterica serovar Typhimurium NCTC12023 and LT2 strains using mouse and human phagocytic and human epithelial cell line models. As macrophage models, the well-characterised mouse cell lines J774, RAW247 were used, along with macrophage cell lines established from CD9 KO and wild type mice. In addition, macrophages derived from THP-1 cells were used as a human cell line model. The human epithelial cell line HEC-1- B and HeLa were similarly investigated. Assays were developed to assess infection by flow cytometry (FACS) analysis in addition to conventional microscopy. Additionally, the role of syntenin, a CD63 tetraspanin partner protein, on Salmonella infection of HeLa cells was investigated. Different patterns of tetraspanin expression were observed on the cell lines by flow cytometry, with CD9 and CD81 generally highly expressed. Interestingly, tetraspanin CD63 showed high expression in human THP-1 derived macrophages, but was present at very low levels on mouse macrophage cell lines. Salmonella expressing green and red fluorescent protein were also successfully generated to facilitate the investigation of infection. Pre-treatment of mouse macrophage cells with anti-CD9, anti-CD81 monoclonal antibody (mAb) significantly decreased Salmonella infection. Comparable results were obtained using both immunofluorescence microscopy and FACS analysis. Anti-CD63 mAbs had no effect, which is likely to relate to the low expression of the tetraspanin by these cells. By contrast, anti-CD63 mAb inhibited infection of human THP1 derived macrophages, whereas anti-CD9 mAb had no effect, which is similar to previously reported findings for human MDM (Hassuna, PhD thesis, University of Sheffield, 2010). In this study, no significant effect on Salmonella infection of epithelial cell lines HEC-1- B and HeLa cells was observed on pre-treatment with individual tetraspanin-specific mAbs. This suggests less involvement of tetraspanins in Salmonella infection of non-phagocytic cells. Pre-treatment of J774 cells with soluble recombinant versions of human or mouse CD9- EC2 showed no effect on Salmonella association. This difference in activity between mAb and EC2s may indicate that Salmonella binding to J774 cells is not reliant on TEMs and the antibodies have a different mechanism of action here. Differences in the infectivity of the CD9KO and wild type mouse macrophage cell lines were noted, although this varied depending on the virulence of the Salmonella strain. CD9 KO cells also showed some differences in expression of other tetraspanins at protein, but not mRNA level as determined by microarray analysis. HeLa cells stably knocked down for syntenin expression were more susceptible to Salmonella infection than control cells; however, no changes were observed in early stages of Salmonella binding. The effects of syntenin KD could relate to alternative syntenin partner proteins such as syndecan. This study suggests a prominent role for tetraspanin CD9 and other tetraspanins in Salmonella infection of mouse macrophages and confirms a role for tetraspanins in infection of human macrophages. The different effects observed between cell lines when targeting tetraspanins could be related to differences in bacterial types, strains and mode of uptake, as well as differences in tetraspanin expression