Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

Staphylocoagulase-binding region in human prothrombin

A staphylocoagulase-binding region in human prothrombin was studied by utilizing several fragments prepared from prothrombin by limited proteolysis. Bovine prothrombin, prethrombin 1, prethrombin 2, and human diisopropylphosphorylated alpha-thrombin strongly inhibited formation of the complex ("staphylothrombin") between human prothrombin and staphylocoagulase, but bovine prothrombin fragment 1 and fragment 2 had no effect on the complex formation, indicating that the binding region of human prothrombin for staphylocoagulase is located in the prethrombin 2 molecule. To identify further the staphylocoagulase-binding region, human alpha-thrombin was cleaved into the NH2-terminal large fragment (Mr = 26,000) and the COOH-terminal fragment (Mr = 16,000) by porcine pancreatic elastase. Of these fragments, the COOH-terminal fragment, which includes Asn-200 to the COOH-terminal end of the alpha-thrombin molecule, partially inhibited the complex formation between staphylocoagulase and human prothrombin. In contrast, the NH2-terminal large fragment did not show any inhibitory effect on the staphylothrombin formation. These results suggest that the staphylocoagulase interacts with human prothrombin through the COOH-terminal region of alpha-thrombin B chain. Other plasma proteins, factor X, factor IX, protein C, protein S, protein Z, all of which are structurally similar to prothrombin, did not inhibit the staphylothrombin formation at all, indicating that a specific interaction site with staphylocoagulase is contained only in the prothrombin molecule.     

Activation of human prothrombin by stoichiometric levels of staphylocoagulase

The activation of human prothrombin by the bacterial protein staphylocoagulase proceeds via the formation of a very stable equimolar complex. Unmasking of the active center in the prothrombin moiety of the complex is not caused by limited proteolysis. The kinetics of activation of human prothrombin by staphylocoagulase has been studied. The second order rate constant at pH 7.5, 37 degrees C, is 3.3 X 10(6) M-1 S-1. This reaction rate is close to reported diffusion-controlled rates of protein-protein interaction. The dissociation constant of the complex was too low to be measurable. From the kinetic data it is assumed that the first order rate constant for dissociation is orders of magnitude less than 10(-5) S-1. However, dissociation of the complex did occur in the presence of sodium dodecyl sulfate. Equimolar amounts of staphylocoagulase protect human thrombin, but not human factor Xa and bovine thrombin, against inactivation by antithrombin III. From these findings we postulate that tertiary structural changes in the thrombin region of prothrombin caused by a highly specific interaction between staphylocoagulase and that region unmask the active site.     

Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+) and Asc10(-)E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin

Staphylocoagulase with a molecular weight of 64,000 and subspecies ranging in molecular weight from 36,000 to 64,000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr = 64,000) from strain st-213 formed an active complex, "staphylothrombin," with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37 degrees C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H]diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of alpha-thrombin was found. The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of alpha-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl-p'-guanidinobenzoate showed 0.95 +/- 0.2 mol of active site/mol of enzyme.     

Development of a mouse model of induced Staphylococcus aureus infective endocarditis

We developed a mouse model of Staphylococcus aureus infective endocarditis to evaluate the efficacy of experimental antibacterial compounds for this disease. Experimental infective endocarditis was produced in CD1 mice by intravenous challenge with approximately 6 log10 colony-forming units (CFU) of methicillin-sensitive (MSSA) SA-3529 or -resistant (MRSA) SA-2015 S. aureus 1 d after aortic valve trauma. Valve trauma was produced by introduction of an indwelling 32-gauge polyurethane catheter into the aortic valve via the left carotid artery. Histologic examination of MSSA- and MRSA-infected and catheterized aortic valve sections revealed neutrophilic inflammation and vegetative bacterial colonies encapsulated within fibrin along the aortic valves 1 d after infection. The MSSA or MRSA endocarditis was determined to be catheter-dependent based on catheterized mice exhibiting heart bacterial counts 4 orders of magnitude greater than those seen for noncatheterized mice. The model was validated by using a 3-d regimen of vancomycin at exposures comparable to human dosing (500 microg x h/ml). Vancomycin treatment produced statistically significant reductions of 3.4 and 3.1 log10 CFU/heart for MSSA and MRSA, respectively, relative to controls. This mouse model of endocarditis shows promise in evaluating the predictive efficacy of antibiotics for S. aureus infective endocarditis.     

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.     

Diffusion of ofloxacin in the endocarditis vegetation assessed with synchrotron radiation UV fluorescence microspectroscopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique i.v. injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510-560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.     

Diffusion of Ofloxacin in the Endocarditis Vegetation Assessed with Synchrotron Radiation UV Fluorescence Microspectrocopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique IV injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510–560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.     

The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase

Staphylococcus aureus encodes a secreted von Willebrand factor-binding protein (vWbp) of 482 amino acids. The N-terminal part of this protein is homologous to staphylocoagulase and therefore we investigated whether vWbp has coagulating activity. Recombinant vWbp was shown to coagulate human and porcine plasma efficiently, but was less active against plasma from other species. The coagulation efficiency was concentration dependent, and could be inhibited by specific antibodies against vWbp. Furthermore, the species-specific coagulation by vWbp depended on the interaction with prothrombin. This interaction also resulted in specific cleavage of vWbp, releasing the C-terminal part from the coagulating domain.     

Bacterial endocarditis of the mitral valve associated with annual calcification.

Bacterial endocarditis, caused mainly by Staphylococcus aureus, was found at autopsy in five patients who had a calcified posterior mitral valve annulus. Clincopathologic correlation indicates that the infection should be suspected in elderly patients with a calcified mitral annulus, the murmur of mitral insufficiency, fever, anemia, polymorphonuclear leukocytosis and a positive blood culture, regardless of evidence of peripheral embolism or of another disease that could cause the last four features. Pertinent pathologic findings are a calcified mitral valve annulus, vegetations of bacterial endocarditis towards the base of the posterior leaflet associated with leaflet perforation and an annulus abscess, and no other valvular disease. The infection may develop on the atrial aspect of a leaflet ulcerated by the calcium mass or may begin on its ventricular aspect, subsequently perforating the leaflet and infecting its atrial surface.     

Experimental endocarditis model of methicillin resistant Staphylococcus aureus (MRSA) in rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.     

Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis

Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n?=?160) and patients with infective endocarditis (n?=?40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P?>?0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P?=?0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P?>?0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis.     

Reverse-vaccinology strategy for designing T-cell epitope candidates for Staphylococcus aureus endocarditis vaccine

Staphylococcus aureus is an opportunistic pathogen causing various inflammatory diseases from skin and tissue local infections, to serious life threatening infections including endocarditis. Experimental models for endocarditis demonstrated that virulence factors of S. aureus, that are very important in infection of heart vegetations, are surface proteins which promote bacterial adherence. Until now, efforts to develop effective vaccines against S. aureus were unsuccessful, partly due to the fact that different vaccine formulations have targeted mainly B-cell immunity. Reverse vaccinology is applied here, in order to identify potential vaccine epitope candidates. The basic epitopes prediction strategy relied on detection of a common antigenic 9-mer epitope meant to be able to stimulate both the B-cell and T-cell mediated immunity. Ten surface exposed proteins were chosen for antigenicity testing. Using a web-based system, five T-cell epitopes corresponding to fibronectin binding protein A (FDFTLSNNV and YVDGYIETI), collagen adhesin (FSINYKTKI), serine-rich adhesin for platelets (LTFDSTNNT) and elastin binding protein (FAMDKSHPE) were selected as potential vaccine candidates. Epitopes sequences were found to be conserved among the different S. aureus genomes screened from NCBI GenBank. In vitro and in vivo immunological tests will be performed in order to validate the suitability of the epitopes for vaccine development.     

Staphylococcus aureus cholecystitis: a report of three cases with review of the literature

Infection of the hepatobiliary system is most commonly due to enteric bacteria. We report three unusual cases of acute cholecystitis in which Staphylococcus aureus was the primary pathogen. Infection of the gallbladder with this organism has been rarely described and may be associated with gallstones and obstructive disease as well as acalculous cholecystitis in the setting of staphylococcal bacteremia and endocarditis. Two of our patients had multiple chronic medical conditions and were infected with oxacillin-resistant S. aureus (ORSA) suggesting nosocomial acquisition. Including our cases with a review of the literature, three of nine reports of S. aureus cholecystitis were associated with infectious endocarditis. Thus, the finding of S. aureus cholecystitis with bacteremia is rare and should prompt an investigation for a possible endovascular focus of infection.     

Isoalantolactone protects against Staphylococcus aureus pneumonia

Staphylococcus aureus is a versatile pathogen that can cause life-threatening infections. The growing emergence of methicillin-resistant S.?aureus strains and a decrease in the discovery of new antibiotics warrant the search for new therapeutic targets to combat infections. Staphylococcus aureus produces many extracellular virulence factors that contribute to its pathogenicity. Therefore, targeting bacterial virulence as an alternative strategy to the development of new antimicrobials has gained great interest. α-Toxin is a 33.2-kDa, water-soluble, pore-forming toxin that is secreted by most S.?aureus strains. α-Toxin is essential for the pathogenesis of pneumonia, as strains lacking α-toxin display a profound defect in virulence. In this report, we demonstrate that isoalantolactone (IAL), a naturally occurring compound found in Inula helenium (Compositae), has no anti-S.?aureus activity as per MIC evaluation in vitro. However, IAL can markedly inhibit the expression of α-toxin in S.?aureus at very low concentrations. Furthermore, the in vivo data indicate that treatment with IAL protects mice from S.?aureus pneumonia.     

Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB,the serine-aspartate repeat protein SdrE and protein A

The ability of Staphylococcus aureus cells to induce platelet aggregation has long been recognized. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial receptors required remains poorly understood. Using genetic manipulation, this study for the first time provides clear evidence that several S. aureus surface proteins participate in the inter-action with platelets. Mutants of S. aureus strain Newman lacking one or more surface proteins were tested for their ability to stimulate platelet aggregation. This approach was complemented by the expression of a number of candidate proteins in the non-aggregating Gram-positive bacterium Lacto-coccus lactis. S. aureus-induced aggregation was monophasic and was dependent on the platelet receptor GPIIb/IIIa. The fibrinogen-binding proteins, clumping factors A and B and the serine-aspartate repeat protein SdrE could each induce aggregation when expressed in L. lactis. Although protein A expressed in L. lactis was not capable of inducing aggregation independently, it enhanced the aggregation response when expressed on the surface of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.     

Noninvasive optical imaging of staphylococcus aureus bacterial infection in living mice using a Bis-dipicolylamine-Zinc(II) affinity group conjugated to a near-infrared fluorophore

Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as light-emitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is a need for exogenous synthetic probes that can selectively target bacteria. The focus of this study is a fluorescent imaging probe that is composed of a bacterial affinity group conjugated to a near-infrared dye. The affinity group is a synthetic zinc (II) coordination complex that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5 x 10 (7) cells) in a mouse leg infection model using whole animal near-infrared fluorescence imaging. Region of interest analysis showed that the signal ratio for infected leg to uninfected leg reaches 3.9 +/- 0.5 at 21 h postinjection of the probe. Ex vivo imaging of the organs produced a signal ratio of 8 for infected to uninfected leg. Immunohistochemical analysis confirmed that the probe targeted the bacterial cells in the infected tissue. Optimization of the imaging filter set lowered the background signal due to autofluorescence and substantially improved imaging contrast. The study shows that near-infrared molecular probes are amenable to noninvasive optical imaging of localized S. aureus infection.