Coagulase Activity by Staphylococcus aureus: A Potential Target for Therapy?

Staphylococcus aureus is a leading cause of skin and soft tissue infections, foreign body infections, and infective endocarditis. In case of endovascular infection with S. aureus, higher rates of cardiac valve destruction, embolic complications, severe sepsis, and death occur. The unique capacity of S. aureus to induce clotting has been known for over a century; however, its role in virulence has long been controversial. S. aureus secretes two coagulases, staphylocoagulase and von Willebrand factor binding protein that both activate prothrombin to generate fibrin. A better understanding of the molecular mechanisms as well as the new strategies to target the coagulases have highlighted their importance in S. aureus virulence. Coagulase activity is essential for the formation of S. aureus-fibrin-platelet microaggregates and for the homing of S. aureus to the vascular wall under flow. Absence or inhibition of S. aureus coagulase activity improved outcome in disease models of skin infection, sepsis, catheter infection, and endocarditis. Here, we review how the manipulation of the host's hemostatic system contributes to the disease-causing potential of S. aureus and discuss the S. aureus coagulases as promising targets for novel therapeutic strategies.

Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis.

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights.

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and ß subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/ß2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/ß2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 µM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

Predominant role of host proteases in myocardial damage associated with infectious endocarditis induced by Enterococcus faecalis in a rat model.

Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

The pitfall of coagulase-negative staphylococci: a case of Staphylococcus lugdunensis endocarditis.

We report a case of a 60-year-old woman. She was transferred from a local hospital to our cardiovascular medicine department with a diagnosis of infectious endocarditis due to Staphylococcus lugdunensis. Transthoracic echocardiograph confirmed the presence of large vegetations on the native aortic and mitral valve, and subsequent severe regurgitation due to the aortic and mitral valve destruction. Emergent operation was performed and patient's life was barely rescued. However, S. lugdunensis belongs to coagulase-negative staphylococci, which are generally regarded as relatively avirulent bacterium, the endocarditis caused by S. lugdunensis can be invasive and often resembles endocarditis due to Staphylococcus aureus. Therefore, whenever this organism is found in patients with endocarditis, early surgical treatment of the infected valve should be considered.

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline.

The crystal structure of GcnA, an N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal alpha-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (beta/alpha)(8) TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-beta-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.

Mitral valve surgery with surgical embolectomy for mitral valve endocarditis complicated by septic coronary embolism.

Acute myocardial infarction (AMI) complicated by septic coronary embolism due to active infective endocarditis is rare but usually fatal. We report a case of successful mitral valve surgery with surgical embolectomy in a 27-year-old man with an AMI complicated by septic coronary embolism due to mitral valve endocarditis. A chest radiograph revealed cardiomegaly and marked pulmonary edema. A transthoracic echocardiogram disclosed severe mitral regurgitation with highly mobile vegetations and hypokinesia of the left ventricular apex. The electrocardiographic findings of ST segment elevation in leads V2-4 and elevated cardiac enzyme levels were strongly suggestive of an acute anterolateral AMI. Nevertheless, emergent cardiac surgery was needed without selective coronary angiography because of intractable heart failure and life-threatening ventricular tachyarrhythmia requiring cardiopulmonary resuscitation. A total occlusion of the distal left anterior descending artery caused by embolic vegetation and thrombus, which was incidentally detected intraoperatively, was successfully recanalized by surgical embolectomy and thrombectomy using a direct coronary incision. The mitral valve endocarditis was managed with wide debridement and mechanical valve replacement. Three years after the surgery a follow-up echocardiogram showed no abnormalities of the regional wall, motion in the left ventricle and the patient is living an active life without any complications.

Mitral valve thrombosis and infective endocarditis in a patient with prothrombin mutation.

This report deals with a 25-year-old female patient, who was admitted to a medical centre with complaints of fatigue and arthralgia. A non-homogeneous mass originating from the posterior papillary muscle and reaching the posterior and anterior cusps of the mitral valve was evident on the patient's echocardiogram. Her intractable fever, despite adequate antibiotic therapy, led us to consult with the cardiac surgeons. A mitral valve replacement was performed on the 8th day of admission. The pathological examination of the mitral valve and the vegetative lesion revealed the presence of organized thrombus and infective endocarditis. The patient had a history of oral contraceptive use for one year and at genetic examination we detected a heterozygous prothrombin mutation (G20210A). The association of infective endocarditis with native mitral valvular thrombosis in a case with prothrombin mutation and history of oral contraceptive use encouraged us to share our experience with our colleagues.

Fsr-independent production of protease(s) may explain the lack of attenuation of an Enterococcus faecalis fsr mutant versus a gelE-sprE mutant in induction of endocarditis.

An Enterococcus faecalis gelE insertion disruption mutant (TX5128), which produces neither gelatinase (GelE) nor the cotranscribed (in the wild type) serine protease (SprE), was found to be attenuated in a rat endocarditis model with a significant decrease in the endocarditis induction rate versus wild-type E. faecalis OG1RF (GelE(+), SprE(+)). TX5266, which has a nonpolar deletion in fsrB and, like TX5128, is phenotypically GelE(-) under usual conditions, was also studied; fsrB is a homologue of agrB of staphylococci and participates in regulation of gelE-sprE expression. Unexpectedly, TX5266 approximated wild-type OG1RF in the endocarditis model and was significantly less attenuated than TX5128. This is in contrast to other models which have found fsr mutants to be as or more attenuated than TX5128. Further study found that the fsrB mutant produced very low levels of gelatinase activity after prolonged incubation in vitro versus no gelatinase activity with TX5128 and did not show the extensive chaining characteristic of TX5128. Reverse transcription-PCR confirmed that gelE was expressed in TX5266 at a very low level versus wild-type OG1RF and was not expressed at all in TX5128. Possible explanations for the increased induction of endocarditis by TX5266 versus TX5128 include the production of low levels of protease(s) or some other effect(s) of the inactivation of the E. faecalis fsr regulator. The equivalent ability of OG1RF and its fsr mutant to initiate endocarditis may explain why we did not find naturally occurring fsr mutants, which account for ca. 35% of E. faecalis isolates, unrepresented in endocarditis versus fecal isolates.

Effective treatment of cephalosporin-rifampin combinations against cryptic methicillin-resistant beta-lactamase-producing coagulase-negative staphylococcal experimental endocarditis.

The efficacy of cefazolin or cefpirome alone or combined with rifampin was compared with that of vancomycin alone or combined with rifampin in an experimental model of methicillin-resistant, beta-lactamase-producing, coagulase-negative staphylococcal endocarditis. Phenotypically, the mecA gene-positive strain used in vivo did not exhibit methicillin resistance by the agar dilution or disk susceptibility method but was resistant in vitro (oxacillin MIC, 64 micrograms/ml) by the microtiter dilution method with 2% NaCl supplementation. Macrodilution broth susceptibilities of standard inocula failed to demonstrate cross-resistance of staphylococci to cefazolin (MIC, 8 micrograms/ml) or cefpirome (MIC, 4 micrograms/ml). In vivo, vancomycin and cefpirome had similar activities, and both regimens were more effective than was cefazolin alone. While the MIC of rifampin was low (0.031 micrograms/ml), monotherapy with rifampin resulted in a bimodal distribution of outcomes due to the expected emergence of resistant mutants. The results in vitro of time-kill synergy studies using rifampin in combination with cefazolin or cefpirome varied with the antimicrobial concentrations tested and did not reliably predict activities in vivo of rifampin-beta-lactam combination therapies. Cefpirome, but not cefazolin or vancomycin, in combination with rifampin was synergistic in vivo. Cefpirome in combination with rifampin was more effective than was cefazolin in combination with rifampin. Both cephalosporin-rifampin regimens were significantly more effective than was cephalosporin or vancomycin monotherapy and were as effective as vancomycin combined with rifampin. These data support further evaluation of rifampin-beta-lactam combinations as possible alternative therapies to vancomycin-containing regimens for selected methicillin-resistant coagulase-negative staphylococcal infections.

Virulence of coagulase-deficient mutants of Staphylococcus aureus in experimental endocarditis.

A sensitive rat endocarditis model which employed relatively small inocula (< or = 10(4) cfu) was used to examine the role of coagulase in the pathogenesis of infection. Rats with indwelling, intracardiac catheters were challenged intravenously with three strains of Staphylococcus aureus. The virulence of a coagulase-positive parental strain DU5808 was compared in terms of its ID50 for resected vegetations and catheters to that of two coagulase-negative mutant strains (DU5809 and DU5814) which had undergone site-specific mutagenesis. Confidence intervals of infection rates were calculated and comparisons were performed of weights of infected vegetations, bacterial concentrations in vegetations and early mortality rates. From these virulence parameters, it was concluded that there were no differences in virulence among the three strains. The results from this investigation support the previous findings in mouse models of subcutaneous infection and mastitis, which indicated that coagulase production by S. aureus does not appear to function as a virulence factor. Together, these data refute the longheld belief that coagulase is important in the pathogenesis of infection and indicate that other markers of virulence must be operative in diseases caused by the enduring pathogen S. aureus.

Piperacillin, tazobactam, and gentamicin alone or combined in an endocarditis model of infection by a TEM-3-producing strain of Klebsiella pneumoniae or its susceptible variant.

The efficacy of tazobactam, a beta-lactamase inhibitor, in combination with piperacillin, was studied in vitro and in rabbit experimental endocarditis due to a Klebsiella pneumoniae strain (KpR) producing an extended-spectrum beta-lactamase, TEM-3, or its nonproducing variant (KpS). In vitro, piperacillin was active against KpS (MIC = 4 micrograms/ml, MBC = 8 micrograms/ml with 10(7)-CFU/ml inoculum) but not against KpR (MIC = MBC = 256 micrograms/ml). Tazobactam (1 microgram/ml) restored the activity of piperacillin against KpR (MIC = 2 micrograms/ml, MBC = 4 micrograms/ml). Gentamicin was active against both strains (MIC = 0.25 and 0.5 micrograms/ml for KpS and KpR, respectively). The piperacillin-tazobactam-gentamicin combination was synergistic in vitro. The piperacillin/tazobactam ratio in plasma and in vegetations was always lower than the 4/1 injected dose ratio. In vivo, piperacillin (300 mg/kg of body weight four times a day [QID]) was active against KpS but not against KpR. Tazobactam (75 mg/kg QID) was able to restore the in vivo effect of piperacillin (300 mg/kg QID) against KpR (-3.0 log10 CFU/g of vegetation versus that of controls). Gentamicin (4 mg/kg twice a day [BID]) was active against both strains. Compared with controls, the combination of gentamicin plus piperacillin against KpS (-5.6 log10 CFU/g of vegetation), and the gentamicin-piperacillin-tazobactam combination against KpR (-4.4 log10 CFU/g of vegetation) achieved the greatest decrease in bacterial counts in vegetations and were the only regimens that significantly increased the proportion of sterile vegetations. It is concluded that (i) tazobactam was able to restore the effect of piperacillin against a TEM-3 extended-spectrum Beta-lactamase-producing strain of K. pneumoniae, both in vitro and in a severe experimental infection with high inoculum, when used in a 4/1 piperacillin/tazobactam dose ratio; (ii) gentamicin alone was effective because of the high peak/MBC ratio in plasma; (iii) piperacillin-tazobactam-gentamicin, probably because of the effect of gentamicin in reducing bacterial inoculum in vivo, as stressed by the results obtained by piperacillin-gentamicin against KpS, may be the most effective regimen against KpR.

Retinal embolization from endocarditis.

Arterial emboli, fragmentation of both the arterial and venous blood columns, and bilateral cherry red spot were seen in the fundus of a patient with progressive onset of visual loss. Initially, the patient, a 56-year-old housewife, was thought to have temporal arteritis. Her clinical condition worsened on steroids which was subsequently discontinued when 5 out of 6 blood cultures were reported positive. Showers of microemboli were responsible for her unusual fundus findings.

Abnormalities in heart membranes and myofibrils during bacterial infective cardiomyopathy in the rabbit.

We studied hearts from sham-operated and uninfected catheterized rabbits as well as from rabbits at early and late stages of cardiomyopathy and failure after 3 and 6 days of infection with Streptococcus viridans. No ultrastructural abnormalities or biochemical changes in membrane and myofibrillar activities were seen in 3-day uninfected hearts. In 6-day uninfected hearts there were decreased sarcolemmal M2+ ATPase, Na+-K+ ATPase, adenylate cyclase and calcium binding, microsomal calcium binding and uptake, and myofibrillar Ca2+-stimulated ATPase as well as increased mitochondrial calcium uptake. Slight ultrastructural changes also were apparent in 6-day uninfected hearts. At both early and late stages of infective cardiomyopathy and failure there were varying degrees of depression in sarcolemmal Mg2+ ATPase, Na+-K+ ATPase, adenylate cyclase and calcium binding, microsomal calcium binding, calcium uptake and basal ATPase, and myofibrillar Ca2+-stimulated ATPase activities. However, sarcolemmal Ca2+ ATPase and myofibrillar Mg2+ ATPase activities were decreased only after 6 days of infection. Mitochondrial calcium binding and uptake were increased in early stages but decreased in late stages of disease. Furthermore in infected hearts there were defects in mitrochondrial respiration and phosphorylation. Generalized severe myocardial cell damage involving myofibrils, mitochondria, and the sarcotubular system was seen only in late stages of infection. The results demonstrate impairment of different membrane and contractile protein functions as well as ultrastructural abnormalities in bacterial cardiomyopathic hearts which were absent or of lesser magnitude in hearts with only hypertrophy. The findings reported here suggest to use that there is an association between heart failure and changes in function of cellular components during bacterial infective cardiomyopathy.