(a-d) Representative S. aureus vegetation induced in a murine model of endocarditis. Hematoxylin and Eosin (H&E; a,b) and Gram staining (c,d) are shown with 400× total magnification in panels (b) and (d). (e) A schematic representation of vegetation formation in S. aureus endocarditis including a hypothetical mechanism for probe localization (AF680-ProT, yellow) after endothelial trauma and induction of bacteremia. Staphylocoagulase (SC) is shown tethering AF680-ProT to fibrinogen/fibrin via D domain interactions. Fibrinogen (inset) has a central E domain flanked by two D domains (dark blue). Schematics of AF680-ProT and SC are also shown. (f-h) Ex vivo fluorescence reflectance imaging (FRI) showed that localization of AF680-ProT is limited to pathogenic S. aureus. White light (f) and fluorescence images show excised aortas with vegetations (g; arrow heads) as well as the location of the suture (arrow). Quantitation of FRI data is given in panel (h) for the controls (either no bacteria or coagulase-negative S. epidermidis) and for three S. aureus strains. TBR: target to background ratio. * indicates P < 0.01. (i-l) Microscopic localization of AF680-ProT correlates with SC-positive immunostaining. Gram stain (i), SC immunohistochemistry (j), in situ hybridization against digoxigenin-SC RNA (DIG-SC) (k) and fluorescence microscopy (l) demonstrate that SC is differentially expressed in growing vegetations and limited to the growing edge. Distance bar equals 50 μm.

(a) A summary diagram showing that prothrombin binding is limited to the NH₂-terminal SC domains (D1-D2) contained in the SC(1-325) fragment, while SC COOH-terminal repeats bind multiple fibrin(ogen) D domains. (b) Fluorescence (top) and white light (bottom) images of the native gels for the following samples: SC(1-325) (lane 1), fluorescent labeled prothrombin (lane 2), fragment D (FragD) (lane 3), labeled prothrombin with 1.1-fold SC(1-325) (lane 4), labeled prothrombin with 1.1-fold SC(1-325) and 8-fold molar excess FragD (lane 5), labeled prothrombin and FragD (lane 6). (c) Similar lanes using full-length SC (SC(1-660)) demonstrate the formation of a SC(1-660)•prothrombin•FragD ternary complex (lane 5). (d) Native gel electrophoresis demonstrating that SC binds multiple FragD domains through COOH-terminal repeats on SC. Mixtures of SC(1-660) with increasing molar ratios of FragD are shown in lanes 2-9 (1, 2, 4, 5, 6, 7, 8 and 10-fold molar excess of FragD); samples of SC(1-660) and FragD alone were run in lanes 1 and 10, respectively. (e) Binding of fragment D to fluorescein-labeled SC pseudorepeat-repeat-1. Titrations of the relative increase in fluorescence (ΔF/F_o) of a 5-(iodoacetamido)fluorescein-labeled Cys residue attached to the COOH-terminus of the SC pseudorepeat-repeat-1 peptide at 20 (filled circle), 430 (open circle), and 855 nM (filled triangle) as a function of the total concentration of fragment D ([FragD]_o). Solid lines represent the simultaneous least-squares fit of the quadratic binding equation with K_D 36 ± 8 nM, stoichiometric factor 0.77 ± 0.06 mol of fragment D/mol of peptide, and a maximum fluorescence change of 8.7 ± 0.2 %. Inset: SDS-PAGE of molecular mass standards in kDa (lane 1), protein-stained gel (4 μg, lane 2), and fluorescence of the same gel (lane 3).

Non-invasive fluorescence molecular tomography fused to computed tomography (FMT-CT) of mice infected with three different pathogenic S. aureus groups (a, Tager 104; b, Xen8.1; c, Xen29), and two control groups injected with either AF680-ProT but no bacteria (d) or with a coagulase-negative S. epidermidis strain (e). (f) Absolute fluorochrome concentration in vegetations quantified by FMT-CT. Data are mean ± SEM, n = 6 per group, * P < 0.01.

Non-invasive fluorescence molecular tomography fused to computed tomography (FMT-CT) of mice infected with (a) strain Newman or (b) Newman rendered genetically deficient in coagulases SC and VWbp. (c) Absolute fluorochrome concentration in vegetations quantified by FMT-CT. Data are mean ± SEM, n = 6 per group,* P < 0.01 (d) Kaplan-Meier curves comparing the survival of mice infected with Newman (blue line) or coagulase-deficient Newman (red line), n =10-13 per group, median survival 2.1 versus 4.3 days, P < 0.0001.

(a,b) Molecular model of Cu⁶⁴-DTPA in the active site of prethrombin 2 (Pre2; yellow) bound to SC (1-325)(violet). DTPA atoms are shown as color-coded spheres (carbon, green; oxygen, red; nitrogen, blue; sulfur, yellow and copper, orange). The two views are related by a 90-degree rotation around the vertical axis. The DTPA moiety was essentially solvent exposed, and moved freely within a region spanned by the 60-, 99- and 148-loops on thrombin. The model indicated that the charged groups on these loops would not affect ⁶⁴Cu(II) ion coordination, and this was confirmed by the high percentage of ⁶⁴Cu (89-95% molar ratio) incorporation by the DTPA-ProT recipient. (c-f) Representative CT (c, long axis; d, short axis) and PET-CT images (e,f) after injection of Cu⁶⁴-DTPA-ProT. The inserted suture is denoted by an arrow, the aortic valve by an asterisk, and an arrow head highlights the vegetation. (g,j) Autoradiography and bioluminescence images of excised aortas from a mouse that received Cu⁶⁴-DTPA-ProT and S. aureus Xen8.1 (g,h) and from a “no bacteria” control (i,j). (k) Correlation of probe uptake (autoradiography) with bacterial load in vegetation (bioluminescence).