Abstract : Streptococcus pneumoniae is the most common cause of otitis, sinusitis, pneumonia, sepsis and meningitis. Recently, pili were identified at the surface of S. pneumoniae and were proposed to play a role in the initial step of host tissues colonisation. Six genes are involved in the pilus formation. Three of them encode structural proteins named pilins (RrgA, RrgB and RrgC), the three others encode specific enzymes called sortases, which catalyse the covalent association of the pilins (SrtC-1, SrtC-2 and SrtC-3). Pilus formation models have been proposed based on genetic studies, but no biochemical data explaining precisely the molecular process of pilus formation is yet available. The individual study of each pilin led to the identification of stabilising Lys-Asn intramolecular bonds in each protein. Moreover, the cristallographic structure of RrgA and RrgB provided precious informations regarding the adhesive properties as well as the mechanism of pilus assembly. Since the role of each sortase remains unclear, our aim was to elucidate, step by step, the molecular mechanisms involved in the pilus biogenesis. Therefore, we have developed a co-expression plateform allowing the production of the pilins subunits together with sortases in E.coli. This system allowed to decipher the substrate specificity of the sortases, to generate covalent pilin/pilin complexes, as well as pilin/sortase complexes and thus provided key information towards the understanding complex macromolecular process.