SUMMARY Systemic dissemination of microbial pathogens permits microbes to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to bacterial infections. Dissemination is a critical stage of disease progression by the Lyme spirochete, Borrelia burgdorferi. However, many mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular surfaces in the face of the shear forces present in flowing blood. Using real-time microscopic imaging of the Lyme spirochete in living mice we previously identified the first bacterial protein (B. burgdorferi BBK32) shown to mediate vascular adhesion in vivo. Vascular adhesion is also dependent on host fibronectin (Fn) and glycosaminoglycans (GAGs). In the present study, we investigated the mechanisms of BBK32-dependent vascular adhesion in vivo. We determined that BBK32-Fn interactions (tethering) function as a molecular braking mechanism that permits the formation of more stable BBK32-GAG interactions (dragging) between circulating bacteria and vascular surfaces. Since BBK32-like proteins are expressed in a variety of pathogens we believe that the vascular adhesion mechanisms we have deciphered here may be critical for understanding the dissemination mechanisms of other bacterial pathogens.

Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca 2؉ , as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, 45 binding plays a pivotal role in the pathogenesis of leptospirosis.Leptospira spp. are spirochetes, including the pathogenic species Leptospira interrogans as well as the saprophytic species Leptospira biflexa. Leptospirosis, a zoonotic disease caused by Leptospira spp., is widely distributed in developing countries and has reemerged in the United States (1). The severe form of leptospirosis, Weil's syndrome, includes an acute febrile illness associated with multiorgan damage, such as liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (2), with a mortality rate up to 15% if not treated (3). Several virulence factors of this organism have been identified, including the sphingomyelinases, serine proteases, zincdependent proteases, collagenase (4), LipL32 (5), lipopolysaccharide (6), a novel factor H, laminin-and fibronectin-binding protein (Lsa24 or Len) (7-9), Loa22 (10), and Lig (leptospira immunoglobulin-like) proteins (11-13).Lig proteins, which include LigA and LigB, possess bacterial immunoglobulin-like (BIg) 3 domains with 90-amino acid tandem repeats. Both proteins have identical N-terminal sequences of 630 amino acids, but their C termini are variable (11-13). LigB also encodes a C-terminal, nonrepeat domain with 771 amino acid residues (11, 12). LigA and LigB may serve as microbial surface components recognizing adhesive matrix molecules that allow pathogenic Leptospira to bind to host extracellular matrix components, such as fibronectin (Fn), fibrinogen, laminin, and collagen (14 -16). Lig proteins may also serve as possible vaccine candidates and/or as diagnostic antigens (12,17,18), and their expression is regulated by osmolarity (19). A high affinity Fn binding region of LigB, designated LigBCen2, contains 152 amino acids that include part of an immunoglobulin-like domain and a nonrepeated region (15) (Fig. 1A).Calcium plays a pivotal role in bacterial physiological activities, such as cell cycle, cell division (20), competence (21), pathogenesis (22), signal transduction (23), and motility and chemotaxis (24,25). Apart from these functions, it is also known that host-pathogen interactions of some bacteria are affected by calcium (26,27). Several types of Ca 3 The abbreviations used are: BIg, bacterial immunoglobulin-like; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; EDS, energy-dispersive spectrometry; ICP-OES, inductively coupled plasma optical emission spectrometry; Fn, fibronectin; NTD, N-terminal domain; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; MOPS, 4-morpholinepropan...

Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (KD = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7'-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7'-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7'-13 and LigBCen7'-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction.