The Role of Tetraspanins in Pseudomonas aeruginosa Adherence to Human Cells

Infection of host tissue is commonly initiated by adherence of pathogenic bacteria, such as Pseudomonas aeruginosa, to cell surface membrane proteins, sugars or lipids. One class of proteins known to play a major role in cellular processes, such as adhesion are tetraspanins, specifically CD9, CD81 and CD151. Prevention of adhesion could provide a new therapeutic strategy for treating infectious diseases. This work aims to investigate the potential of using recombinant tetraspanin extracellular domains (EC2), anti-tetraspanin antibodies and CD9 EC2-derived peptides as anti-adhesives in treating P. aeruginosa infections. Also, to create a new higher throughput method to measure bacterial adhesion using flow cytometry. Expression profiles of different tetraspanins in human epithelial cell lines, A549, HEC1B and keratinocyte HaCaT cells were visualised using immunofluorescent microscopy and quantified using flow cytometry. Monolayers of adherent cells were pre-treated with the tetraspanin reagents (EC2: GST recombinant proteins, anti-tetraspanin antibodies and CD9 EC2-derived peptides), before infection with GFP-labelled P. aeruginosa (PAO1) for 60 minutes. Bacterial adhesion was determined by counting the number of bacteria per infected cells with fluorescence microscopy. Analysis revealed the anti-adhesive properties of these tetraspanins (anti-tetraspanin antibodies and recombinant proteins). In conclusion, a flow cytometry method was developed but was found to be too un sensitive to measure changes in bacterial adhesion. Moreover, anti-tetraspanin antibodies, recombinant proteins and CD9 EC2- derived peptides may have the potential to inhibit adherence of P. aeruginosa. This work has identified a role for tetraspanins in bacteria-host cell adhesion, and the possible use of tetraspanin reagents as therapeutics for treating P. aeruginosa infection.