Structural and Functional Role of the Surface-Exposed Loops of Ail During Complement-Mediated Evasion by Y. pestis

Abstract

Yersinia pestis, the causative agent of plague, promotes its proliferation in the host by expressing various bacterial proteins that inhibit the host innate immune system. One of these is attachment invasion locus (Ail), which is an eight-stranded β-barrel outer membrane protein with four surface exposed extracellular loops. Ail is essential for Y. pestis pathogenesis and proliferation as it recruits host serum proteins to inhibit the complement-dependent bacteriolysis and inflammatory response, and regulates host cell attachment and delivery of Yersinia outer proteins to host tissue. Recently, we demonstrated that Y. pestis can also recruit vitronectin (Vn) from the human serum in Ail-dependent manner. Vn is a 459-residue multi-domain protein that interacts with various molecules to promote cell adhesion and migration, and inhibits formation of the membrane attack complex, indicating that the interaction between Ail and Vn may be responsible for promoting Y. pestis survival and pathogenesis. Notably, previous works have indicated that serum proteins such as Vn are recruited by Ail via its extracellular loops. Thus, we hypothesized that Ail's extracellular loops may also be responsible for binding to Vn. To characterize this interaction, amino acids in each extracellular loop of Ail were substituted for alanine to individually characterize the loops for Vn binding. The structure and function of each Ail loop mutant was characterized in a native like environment inside membrane (detergent micelle and nanodiscs) by solution state NMR and ELISA binding assays. These studies identify specific extracellular loops of Ail that are important for binding to Vn, which will guide future developments of therapeutics aimed at combating Y. pestis infections.