Structural and Mechanistic Insight into how Pathogenic Bacteria Assemble Adhesive Surface Pili via Isopeptide Bonds

Abstract

Infections caused by antibiotic-resistant bacterial pathogens are a significant threat to public health. Many species of Gram+ bacteria display pili (hair-like filaments that protrude from the cell wall) to form biofilms and to adhere to host tissues and cells. Pili are constructed on the cell surface by sortase pilin polymerases, which link pilin subunits together via isopeptide bonds. Although pilus assembly has been studied at a cellular level, the mechanism of sortase-catalyzed isopeptide bond formation is poorly understood. Here we report the integrated use of x-ray crystallography, NMR, biochemistry, biophysics and cellular biology methods to explore the mechanism of assembly of the archetype SpaA-pilus from Corynebacterium diphtheriae. The crystal structure of the pilin polymerase, SrtA, reveals that it is held in an inactive state by an auto-inhibitory “lid” structure. We demonstrate that targeted mutations introduced into the lid activate the enzyme and enable in vitro pilus assembly. Phylogenetic comparisons, amino acid mutagenesis, NMR and a novel in vitro assay define unique enzyme features required for isopeptide polymerization activity and provide insight into the structure of the reaction intermediate that is formed during repeated polymerization cycles. Finally, we present progress toward determining the solution structure of the basal SpaB pilin subunit, which is required for efficient cell wall anchoring of the completed pilus polymer. The structure and mechanistic studies of SpaB incorporation into the completed pilus provide insight into the mechanism of termination. The pilus assembly reaction studied here is highly conserved and can be important for bacterial virulence, thus the results of this work are broadly applicable and may facilitate the design of pilus assembly inhibitors with useful therapeutic properties.