Staphylococcus aureus ß-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

ß-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of ß-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N ß-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. ß-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of ß-toxin and suggests ß-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Role of vegetation-associated protease activity in valve destruction in human infective endocarditis.

AIMS: Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization. METHODS AND RESULTS: IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor. CONCLUSION: Our data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases.

The impact of penicillinase on cefamandole treatment and prophylaxis of experimental endocarditis due to methicillin-resistant Staphylococcus aureus.

Beta-lactams active against methicillin-resistant Staphylococcus aureus (MRSA) must resist penicillinase hydrolysis and bind penicillin-binding protein 2A (PBP 2A). Cefamandole might share these properties. When tested against 2 isogenic pairs of MRSA that produced or did not produce penicillinase, MICs of cefamandole (8-32 mg/L) were not affected by penicillinase, and cefamandole had a > or =40 times greater PBP 2A affinity than did methicillin. In rats, constant serum levels of 100 mg/L cefamandole successfully treated experimental endocarditis due to penicillinase-negative isolates but failed against penicillinase-producing organisms. This suggested that penicillinase produced in infected vegetations might hydrolyze the drug. Indeed, cefamandole was slowly degraded by penicillinase in vitro. Moreover, its efficacy was restored by combination with sulbactam in vivo. Cefamandole also uniformly prevented MRSA endocarditis in prophylaxis experiments, a setting in which bacteria were not yet clustered in the vegetations. Thus, while cefamandole treatment was limited by penicillinase, the drug was still successful for prophylaxis of experimental MRSA endocarditis.

Bromide-dependent toxicity of eosinophil peroxidase for endothelium and isolated working rat hearts: a model for eosinophilic endocarditis.

Eosinophilic endocarditis is a potentially lethal complication of chronic peripheral blood hypereosinophilia. We hypothesized that eosinophil peroxidase (EPO), an abundant eosinophil (EO) cationic granule protein, promotes eosinophilic endocarditis by binding to negatively charged endocardium, and there generating cytotoxic oxidants. Using an immunocytochemical technique, we demonstrated endocardial deposition of EPO in the heart of a patient with hypereosinophilic heart disease. Because EPO preferentially oxidizes Br- to hypobromous acid (HOBr) rather than Cl- to hypochlorous acid (HOCl) at physiologic halide concentrations, we characterized the Br(-)-dependent toxicity of both activated EOs and purified human EPO towards several types of endothelial cells and isolated working rat hearts. In RPMI supplemented with 100 microM Br-, phorbol myristate acetate-activated EOs, but not polymorphonuclear leukocytes, caused 1.8-3.6 times as much 51Cr release from four types of endothelial cell monolayers as in RPMI alone. H2O2 and purified human EPO, especially when bound to cell surfaces, mediated extraordinarily potent, completely Br(-)-dependent cytolysis of endothelial cells that was reversed by peroxidase inhibitors, HOBr scavengers, and competitive substrates. We further modeled eosinophilic endocarditis by instilling EPO into the left ventricles of isolated rat hearts, flushing unbound EPO, then perfusing them with a buffer containing 100 microM Br- and 1 microM H2O2. Acute congestive heart failure (evidenced by a precipitous decrement in rate pressure product, stroke volume work, aortic output, and MVO2 to 0-33% of control values) ensued over 20 min, which deletion of EPO, Br-, or H2O2 completely abrogated. These findings raise the possibility that EPO bound to endocardial cells might utilize H2O2 generated either by overlying phagocytes or endogenous cardiac metabolism along with the virtually inexhaustible supply of Br- from flowing blood to fuel HOBr-mediated cell damage. By this mechanism, EPO may play an important role in the pathogenesis of eosinophilic endocarditis.

[Determination of creatine kinase and CK-MB in heart failure (author's transl)]. / Bestimmung der Creatinkinase und des Isoenzyms Creatinkinase-MB bei Herzinsuffizienz.

Serum activity of creatine kinase and creatine kinase-MB have been investigated in 129 patients of various etiology in overt heart failure. Elevations in CK-MB were found in 19 patients, most frequently in patients with inflammatory heart disease. We found no correlation between CK-MB activity in serum and the severity of heart failure. CK-MB elevation in patients with chronic heart failure may be interpreted as a sign of progressive as well as regressive processes in the myocardium.

[Effect of heparin on certain indicators of oxidating and energetic processes in experimental endomyocarditis]. / Vliianie geparina na nekotorye pokazateli okislitel'nykh i energeticheskikh protsessov pre eksperimental'nom endomiokardite

The influence of therapeutic dosage of heparin on the content of underoxidized substanses of blood, quantity of ATP, ADP, AMP, creatininphosphate, CPK, and lactatedehydrogenase activity of the heart, liver and kidney tissues was studied in 55 rabbits during horse serum and complex antigen sensitization. Changes in oxidizing and energy processes in experimental endomyocarditis were more prominent in these organs tissues than in blood. Heparin administration increased lactate acid in blood and lactatedehydrogenase activity in the cardiac tissue. Heparin also normalized the increased pyruvic acid level in blood and preserved higher ATP content in tissues. Adenyl system components were balanced in heparinized animals. Heparin influence on oxydizing and energy processes depended upon the basic status of the organism.