Interaction Of Leptospiral Microbial Surface Components Recognizing Adhesive Matrix Molecules With Host Extracellular Matrix
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Adhesion through microbial surface components recognizing adhesive matrix molecules (MSCRAMM) is an essential step of infection for most pathogenic bacteria. Leptospira spp. are pathogenic spirochetes that cause a zonootic disease and express several important virulence factors on their surface including immunoglobulin-like (Ig-like) proteins, LigA and LigB. In this dissertation, both LigA and LigB were discovered as MSCRAMMs to bind to some extracellular matrices such as fibronectin (Fn), elastin, tropoelastin, laminin, and collagen. The Fn binding sites are located on LigBCen, C-terminal unique Ig-like domains and LigBCtv, C-terminal non-Ig-like region. In addition, those Fn-binding fragments can also mediate leptospiral adhesion to host cells. A high affinity Fn-binding site was identified on LigBCen2, and the domains of Fn those contribute to the binding were N-terminal domain (NTD) and gelatin binding domain (GBD). Apart from Fn, LigBCen2 can interact with laminin, collagen, and fibrinogen. Interestingly, LigBCen2 was also found as a calcium-binding protein, and calcium-induced conformational change can assist LigB-NTD interactions. Furthermore, LigBCen2 was found to contained a well folded region, LigBCen2R containing 12th and partial 11th Ig-like domains, and a disorered region, LigBCen2NR. LigBCen2R and LigBCen2NR bind to GBD and NTD, respectively. There would be a disordered to ordered transition on LigBCen2NR upon NTD binding. In addition to LigBCen2R, most of Ig-like domains on LigA (7’-8th, 10th, 11th, 12th, 13th) and LigB (7’-8th, 9th) can bind to GBD. The binding affinity of GBD or MDCK cells become greater if the Lig proteins contain more Ig-like domains especilly including the terminal Ig-like domain (LigA13 or LigB12). It suggests that Lig-GBD interaction is enhanced by multivalency to mediate leptospiral adhesion to host cells. Interestingly, Lig proteins with terminal Ig-like domains (LigAVar7’-13 or LigBCen7’-12) bind GBD with 40-fold greater affinities than it without terminal Iglike domains (LigAVar7’-12 or LigBCen7’-11). The compact structure possessed by LigAVar7’-12 or LigBCen7’-11 instead of LigAVar7’-13 or LigBCen7’-12 suggested the relevance to their strong binding affinities with GBD and host cells. Moreover, the Fn-binding sites of LigBCtv was also identified and located on amino acids 1708-1712, LIPAD containing region, and 15th type III modules of Fn (15F3) is characterized to be the slow and moderate binding partner of LIPAD containing region. LIPAD containing region was proved to be surface exposed and possesses a nacent helix and β-strand structure. Elastin and tropoelastin were also discovered to interact with Ig-like domains of Lig proteins. Interestingly, elastin and tropoelastin can bind to conserved region of Lig, which other ECMs don’t bind. Tropoelastin-Lig interaction is attributed to chargecharge interactions, and ASP341 on 4th Ig-like domain (LigCon4) serves as an imporant role for the binding. The binding of Lig proteins to tropoelastin might be elicited to inhibit elastogenesis, then, to help the leptospiral entry by preventing tissue repair and reorganization. Fibrinogen (Fg), a plasma rich protein can be also associated with several Ig-like domains of Lig proteins. LigBCen2R including partial 11th and full 12th Ig-like domains of LigB, can bind to FgαCC, the C-terminal αC domain of Fg. By binding to LigBCen2R, the RGD motif of FgαCC can be blocked and prevent its further interaction with integrin αIIbβ3 for platelet adhesion and aggregation. LigBCen2R- FgαCC can also inhibit thrombin-induced fibrin clot formation but not influence the binding of plasminogen or tissue plasminogen activator. To sum up, Lig-Fg interaction blocking platelet adhesion, aggregation and clot formation might be the one of the reasons to lead pulmonary hemorrhage in Leptospira infected patients.
dissertation or thesis