Structural study of fimbrial adhesin SafD

Abstract: Salmonellosis is a major manifestation of enteric disease in humans across the world. Salmonella possess multiple fimbriae on their surface and many of which play a vital role in attachment with enterocyte of host tissue to establish and maintain a successful infection. The most common virulence strains of Salmonella assemble on their surface. Salmonella atypical fimbriae (Saf), consisting of the major SafA and minor SafD subunits. The Saf fimbriae, as many other virulence organelles, are assembled via the chaperone/usher pathway. The SafB periplasmic chaperone assists in subunit folding and assembly, while the SafC usher acts as a fibre assembly platform and a secretion channel. The SafA subunit has been shown to form the main shaft of the fimbriae. The assembly of SafA subunits is based on the principle of donor strand complementation (DSC). The subunit has incomplete immunoglobulin (Ig) fold and hence possess a large hydrophobic “acceptor” cleft. In the fibre, each subunit inserts an N-terminal sequence into the acceptor cleft of the neighbouring subunit hence completing the Ig fold with a donor strand. Although SafD has been shown to act as a highly protective antigen against Salmonella enteritidis, the function, structure, and location of SafD in Saf fimbriae are not known. In this study, we expressed SafD fused with the N-terminal donor sequence of SafA (SafD-dsSafA) and analyzed its structure. Our temperature and chemical denaturation experiments demonstrated that SafD-dsc is a stable protein. The successful complementation of SafD with the SafA donor strand strongly suggests SafD is complemented by SafA that in the native fibres. On the other hand, mass spectrometry analysis and modelling revealed that SafD does not contain any own N-terminal sequence that could play role of a donor strand. Hence, in contrast to SafA, SafD cannot form a polymer. Based on these facts, we propose that SafD is situated at the tip of the SafA shaft. Such a special location of SafD suggests that it might play an important role in adhesion or invasion. To check this hypothesis, we aimed at determining a high-resolution structure of SafD, its location on the cell surface of Salmonella, and potential SafD receptors on the host cells. We have obtained 2 Å diffracting crystals of SafD and its structure determination is in progress. The SafD-dscSafA construct might be useful to design novel subunit vaccines and rapid diagnostic kits against most common Salmonella virulence strains.