Induction of cytokines by glucosyltransferases of streptococcus mutans

Production of proinflammatory cytokines is implicated in the pathogenesis of viridans streptococcus-induced alpha-streptococcal shock syndrome and infective endocarditis. Streptococcus mutans, one of the opportunistic pathogens causing infective endocarditis, was reported previously to stimulate monocytes and epithelial and endothelial cells in vitro to produce various cytokines. We found that glucosyltransferases (GTFs) GtfC and GtfD of S. mutans stimulated predominantly the production of interleukin-6 (IL-6) from T cells cultured in vitro. The level of IL-6 but not of tumor necrosis factor alpha in blood was significantly elevated when rats were injected intravenously with S. mutans GS-5, whereas IL-6 was detected at a much lower level when rats were challenged with NHS1DD, an isogenic mutant defective in the expression of GTFs. The serum IL-6 level was elevated in patients with endocarditis caused by different species of viridans streptococci which express GTF homologues. Affinity column-purified GTFs reduced the levels of detectable IL-2 of T cells stimulated by another bacterial antigen, tetanus toxoid. These results suggested that GTFs might modulate the production of Th1-type cytokines and that GTFs of S. mutans play a significant role in stimulating the production of the proinflammatory cytokine IL-6 in vivo.     

Induction of Cytokines by Glucosyltransferases of Streptococcus mutans

Production of proinflammatory cytokines is implicated in the pathogenesis of viridans streptococcus-induced α-streptococcal shock syndrome and infective endocarditis. Streptococcus mutans, one of the opportunistic pathogens causing infective endocarditis, was reported previously to stimulate monocytes and epithelial and endothelial cells in vitro to produce various cytokines. We found that glucosyltransferases (GTFs) GtfC and GtfD of S. mutans stimulated predominantly the production of interleukin-6 (IL-6) from T cells cultured in vitro. The level of IL-6 but not of tumor necrosis factor alpha in blood was significantly elevated when rats were injected intravenously with S. mutans GS-5, whereas IL-6 was detected at a much lower level when rats were challenged with NHS1DD, an isogenic mutant defective in the expression of GTFs. The serum IL-6 level was elevated in patients with endocarditis caused by different species of viridans streptococci which express GTF homologues. Affinity column-purified GTFs reduced the levels of detectable IL-2 of T cells stimulated by another bacterial antigen, tetanus toxoid. These results suggested that GTFs might modulate the production of Th1-type cytokines and that GTFs of S. mutans play a significant role in stimulating the production of the proinflammatory cytokine IL-6 in vivo.     

Glucosyltransferases of viridans group streptococci modulate interleukin-6 and adhesion molecule expression in endothelial cells and augment monocytic cell adherence

Recruitment of monocytes plays important roles during vegetation formation and endocardial inflammation in the pathogenesis of infective endocarditis (IE). Bacterial antigens or modulins can activate endothelial cells through the expression of cytokines or adhesion molecules and modulate the recruitment of leukocytes. We hypothesized that glucosyltransferases (GTFs), modulins of viridans group streptococci, may act directly to up-regulate the expression of adhesion molecules and also interleukin-6 (IL-6) to augment monocyte attachment to endothelial cells. Using primary cultured human umbilical vein endothelial cells (HUVECs) as an in vitro model, we demonstrated that GTFs (in the cell-bound or free form) could specifically modulate the expression of IL-6, and also adhesion molecules, in a dose- and time-dependent manner. Results of inhibition assays suggested that enhanced expression of adhesion molecules was dependent on the activation of nuclear factor kappaB (NF-kappaB) and extracellular signal-regulated kinase and that p38 mitogen-activated protein kinase pathways also contributed to the release of IL-6. Streptococcus-infected HUVECs or treatment with purified IL-6 plus soluble IL-6 receptor alpha enhanced the expression of ICAM-1 and the adherence of the monocytic cell line U937. These results suggest that streptococcal GTFs might play an important role in recruiting monocytic cells during inflammation in IE through induction of adhesion molecules and IL-6, a cytokine involved in transition from neutrophil to monocyte recruitment.     

Activated Human Valvular Interstitial Cells Sustain Interleukin-17 Production To Recruit Neutrophils in Infective Endocarditis

The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4⁺ CD45RA⁻ CD25⁻ CCR6⁺ Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4⁺ T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify the release of neutrophilic contents in situ, which might lead to severe valvular damage.     

Induction of experimental endocarditis by continuous low-grade bacteremia mimicking spontaneous bacteremia in humans

Transient high-grade bacteremia following invasive procedures carries a risk of infective endocarditis (IE). This is supported by experimental endocarditis. On the other hand, case-control studies showed that IE could be caused by cumulative exposure to low-grade bacteremia occurring during daily activities. However, no experimental demonstration of this latter possibility exists. This study investigated the infectivity in animals of continuous low-grade bacteremia compared to that of brief high-grade bacteremia. Rats with aortic vegetations were inoculated with Streptococcus intermedius, Streptococcus gordonii or Staphylococcus aureus (strains Newman and P8). Animals were challenged with 10(3) to 10(6) CFU. Identical bacterial numbers were given by bolus (1 ml in 1 min) or continuous infusion (0.0017 ml/min over 10 h). Bacteremia was 50 to 1,000 times greater after bolus than during continuous inoculation. Streptococcal bolus inoculation of 10(5) CFU infected 63 to 100% vegetations compared to 30 to 71% infection after continuous infusion (P > 0.05). When increasing the inoculum to 10(6) CFU, bolus inoculation infected 100% vegetations and continuous infusion 70 to 100% (P > 0.05). S. aureus bolus injection of 10(3) CFU infected 46 to 57% valves. This was similar to the 53 to 57% infection rates produced by continuous infusion (P > 0.05). Inoculation of 10(4) CFU of S. aureus infected 80 to 100% vegetations after bolus and 60 to 75% after continuous infusion (P > 0.05). These results show that high-level bacteremia is not required to induce experimental endocarditis and support the hypothesis that cumulative exposure to low-grade bacteremia represents a genuine risk of IE in humans.     

Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis

The direct binding of bacteria to platelets is a central interaction in the pathogenesis of infective endocarditis. GspB is a serine-rich, cell wall glycoprotein of Streptococcus gordonii that mediates the binding of this organism to human platelets in vitro. To assess the contribution of this adhesin to the pathogenesis of endocarditis, we compared the virulence of S. gordonii M99 (which expresses GspB) with an isogenic, gspB mutant (PS846) in two rat models of endovascular infection. In the first group of experiments, animals were infected intravenously with M99 or PS846, and sacrificed 72 h later, to assess levels of bacteria within cardiac vegetations, kidneys, and spleens. When inoculated with 10(5)CFU, rats infected with PS846 had significantly lower densities of organisms within vegetations (mean: 3.84 log(10)CFU/g) as compared with M99-infected rats (6.67 log(10)CFU/g; P<0.001). Marked differences were also seen in rats co-infected with M99 and PS846, at a 1:1 ratio. While M99 was found at high levels within vegetations, kidneys and spleens (mean log(10)CFU/g: 6.62, 5.07 and 4.18, respectively) PS846 was not detected within these tissues. Thus, platelet binding by GspB appears to be a major interaction in the pathogenesis of endocarditis due to S. gordonii.     

Insertional inactivation of pac and rmlB genes reduces the release of tumor necrosis factor alpha,interleukin-6, and interleukin-8 induced by Streptococcus mutans in monocytic,dental pulp,and periodontal ligament cells

Streptococcus mutans possesses different cell wall molecules, such as protein of the I/II family, the serotype f polysaccharide rhamnose glucose polymer (RGP), and lipoteichoic acid (LTA), which act as adhesins and modulins, allowing S. mutans to colonize teeth and cause dental caries and pulpitis. We tested several isogenic mutants of S. mutans defective in protein I/II and/or RGP, as well as purified modulins such as protein I/II, RGP, and LTA, for their binding and activation abilities on monocytic, dental pulp (DP), and periodontal ligament (PDL) cells. Our results demonstrate that both protein I/II and RGP play important roles in streptococcal adherence to human monocytic and fibroblastic cells, whereas LTA is only a minor adhesin. In the activation process, the cytokine response elicited is polarized toward a Th1 response which seems principally due to protein I/II and RGP. Even if protein I/II seems to be more efficient in its purified form in triggering cells to release interleukin-8 (IL-8), RGP is the most efficient cytokine-stimulating component in intact bacteria, while LTA plays only a minor role. In cell activation, we showed, by using either cytochalasin D or coated ligands, that internalization of either S. mutans, S. mutans isogenic mutants, or purified ligands is not necessary to trigger cells to release IL-8. We also showed that, besides the implication of monocytes in pulpal inflammation, fibroblast-like cells such as DP and PDL cells are also actively implicated in local inflammation and in the generation of a Th1 response after stimulation with S. mutans cells or antigens.     

Rat model of experimental endocarditis.

A simple model of infective endocarditis was produced in rats. With the aid of a guide wire, polyethylene catheters were passed into the left ventricle through the right carotid artery of Sprague-Dawley rats weighing 300 to 350 g. A volume of 1 ml of an overnight culture of Streptococcus mitis, Staphylococcus aureus, or Streptococcus faecalis was intravenously injected 1 to 2 days after catheterization. Bacterial titers of Streptococcus mitis in vegetations were about 10(4)-fold greater than in other tissues. Blood cultures were always positive after 6 h. Mortality was 19% at 1 week and 82% at 2 weeks. Catheters were pulled 24 h after infection, and vegetation titers of greater than 7.0 log10 colony-forming units per g were sustained at 5 days. In intravenously infected rats without catheters, blood and tissues were sterile after 3 to 5 days. With Staphylococcus aureus, vegetations had greater than 9.0 log10 colony-forming units and with Streptococcus faecalis 8.8 +/- 0.3 log10 colony-forming units per g at 2 days. The rat model of infective endocarditis should prove to be suitable for further pathological and therapeutic studies.     

Mycobacterium fortuitum prosthetic valve endocarditis: a case for the pathogenetic role of biofilms

BackgroundProsthetic valve endocarditis presents unique challenges for both diagnosis and treatment. A potential role of biofilm has been hypothesized in the pathogenesis of these infections.MethodsA patient with infective endocarditis involving a stentless (Freestyle) porcine prosthetic aortic valve with annular abscess and paravalvular leak 8 months after implantation is reported.ResultsThe infected valve did not show vegetations or perforations, but histiocytic inflammation was seen along the endocardial surfaces of the valve. Auramine–rhodamine staining revealed many acid-fast organisms associated with the inflammation. There was also an acellular matrix material with ultrastructural features of biofilm. Blood cultures grew Mycobacterium fortuitum, a biofilm-associated microbe.ConclusionsThe role of biofilm in prosthetic valve endocarditis is discussed. The importance of microscopy for prosthetic valves, even when no vegetations are present, is highlighted along with correlation of pathologic findings with culture results.     

Role of the Vegetation in Experimental Streptococcus viridans Endocarditis

This study examines the role of the vegetation in catheter-induced experimental endocarditis in predisposing to bacterial colonization of cardiac valves and in influencing the course of the disease and response to penicillin therapy. Platelet-fibrin vegetations developed at areas of valvular trauma and were colonized when Streptococcus viridans were injected intravenously. Pretreatment with warfarin prevented vegetation formation, but animals still developed endocarditis at the same rate after injection of 10(6)S. viridans. The course of the disease in anticoagulated animals was more explosive, as determined by a more rapid rise in fever and level of bacteremia. Mean survival was shorter in anticoagulated rabbits (7 versus 12.7 days). Large vegetations containing 10(9)S. viridans/g were found in control animals, whereas anticoagulated rabbits developed only microscopic deposits. Large vegetations required a longer duration of penicillin therapy to sterilize than the infected valves of the anticoagulated group (7 versus 3 days). Therefore, a preformed platelet-fibrin deposit is not a prerequisite for bacterial colonization of cardiac valves. After infection, the vegetation is an important factor in determining the subacute course of disease and resistance to penicillin therapy.     

Molecular diagnosis of infective endocarditis by PCR amplification and direct sequencing of DNA from valve tissue

We used broad-range eubacterial PCR amplification followed by direct sequencing to identify microbial pathogens in heart valve material from 29 patients with histologically confirmed infective endocarditis and 23 patients free of infective endocarditis. Microorganisms cultured by conventional techniques matched those identified by PCR in 21 cases. PCR alone identified the causative agent in three cases (Streptococcus bovis, Staphylococcus cohnii, and Coxiella burnetii), allowing better patient management. PCR corrected the initial bacteriological diagnosis in three cases (Streptococcus bovis, Streptococcus mutans, and Bartonella henselae). Among the 29 cases of histologically confirmed infective endocarditis, PCR findings were positive in 27 cases and were consistent with the bacterial morphology seen at Gram staining (26 cases) or with the results obtained by immunohistologic analysis with an anti-C. burnetii monoclonal antibody (one case). In two other cases of histologically confirmed infective endocarditis, PCR remained negative in a blood culture-negative case for which no bacteria were seen at histological analysis and in one case with visualization of cocci and blood cultures positive for Enterococcus faecalis. Ten clinical diagnoses of possible infective endocarditis were ruled out by histopathological analysis of the valves and subsequently by PCR. PCR was negative in 13 of the 14 patients in whom infective endocarditis was rejected on clinical grounds; the other patient was found to have Coxiella burnetii infective endocarditis on the basis of PCR and histopathological analysis and was subsequently included in the group of 29 definite cases. In total, PCR contributed to the diagnosis and management of infective endocarditis in 6 of 29 (20%) cases.     

Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus.

The adherence characteristics in vivo and virulence of two isogenic strains of Staphylococcus aureus differing in fibronectin binding were compared in a rat model of catheter-induced infective endocarditis. No differences were found between the two strains. The results strongly point to the multifactorial nature of bacterial adherence to damaged heart valves and suggest that other binding functions can compensate for the lack of fibronectin binding in S. aureus.     

Effects of molecular weight of dextran on the adherence of Streptococcus sanguis to damaged heart valves.

Dextran-producing streptococci such as Streptococcus sanguis are the organisms most frequently associated with infective endocarditis in humans. A series of experiments was designed to study how the molecular weight of dextrans affects the adherence of an endocarditis strain of S. sanguis to canine heart valves covered with platelets and fibrin. The data indicated that this adherence was dependent on dextrans of high molecular weight, such as dextran T-2000 or glucans isolated from S. sanguis or S. mutans. The adherence properties of the strain studied were not modified by prior exposure of the bacterial cells of valve leaflets to high-molecular-weight dextrans. Preexposure of bacterial cells or valve leaflets to low-molecular-weight dextrans decreased their adherence. Low-molecular-weight dextrans interfered with adherence of dextran-positive strains to damaged heart valves.     

Multi-valvular endocarditis

Objective   Seventy-seven cases of native valve infective endocarditis as determined by the Duke criteria, were reviewed to determine the incidence and clinical features of multi-valvular endocarditis.
Methods   Fourteen of 77 patients (18%) had multi-valvular endocarditis most commonly involving the mitral and aortic valves. Staphylococcus aureus (43%) and viridans streptococci (36%) were the most common organisms causing multi-valvular endocarditis.
Results   Definite or probable vegetations were found in 50% of the patients by two-dimensional transthoracic echocardiograph and/or transesophageal echocardiograph, and possible vegetations were detected in 21%. The overall mortality in our series was 21%; 29% underwent valve replacement and 50% were treated medically. The major complications of multi-valvular endocarditis were congestive heart failure (64%), acute renal failure (50%), embolic events (21%), and splenic abscess/infarcts (21%).
Conclusions   Our data suggests complications of multi-valvular endocarditis, compared with uni-valvular endocarditis are similar except for heart failure. Heart failure is statistically more common in multi-valvular endocarditis ( P 0.002).     

Enhancement of experimental bacteremia and endocarditis caused by dysgonic fermenter (DF-2) bacterium after treatment with methylprednisolone and after splenectomy.

The dysgonic fermenter-2 bacterium is a newly recognized fastidious gram-negative bacillus that causes bacteremia and sometimes endocarditis in immunocompromised persons after they are bitten by dogs. To develop an experimental model of this infection, we placed polyethylene catheters across the aortic valves of New Zealand white rabbits, which were inoculated intravenously the next day with dysgonic fermenter-2 bacteria. After 1 week, the rabbits were killed and the endocardial vegetations were homogenized for quantitative culture. Large inocula (1.3 X 10(10) to 2.1 X 10(10) viable bacteria) were required to produce infected vegetations. All infected rabbits had negative blood cultures at the time of autopsy and most developed serum agglutinins against dysgonic fermenter-2 bacteria. Three daily injections of methylprednisolone (30 mg/kg), starting the day before inoculation, significantly increased the incidence of endocarditis and the number of bacteria per gram of infected vegetation (P less than 0.05). Treatment with methylprednisolone prolonged the initial bacteremia and caused significant increases in the numbers of bacteria per gram of blood, spleen, and liver compared with those of untreated controls (P less than 0.05). Rabbits that had previously undergone splenectomy showed prolongation of the initial bacteremia but no significant increase in the incidence of infected vegetations. These results showed that the dysgonic fermenter-2 bacterium is a pathogen that causes endocarditis in rabbits but that it requires a large inoculum and produces blood culture-negative infections. Treatment with methylprednisolone enhances infection by prolonging the initial bacteremia and probably by diminishing bactericidal activity in the vegetations.     

Protective role of complement in experimental Escherichia coli endocarditis.

Fourteen strains of Escherichia coli were tested for ability to cause infective endocarditis in rabbits prepared by prior placement of an intracardiac catheter. Strains that were resistant to the bactericidal action of serum caused E. coli endocarditis in 91.4% of rabbits, whereas serum-sensitive strains usually failed to cause persisting infection (11.3% infected, P less than 0.001). Although serum-sensitive E. coli lodged on heart valves within 1 h after intravenous injection, they survived less than 24 h in most normal rabbits. In contrast to normals, all five C6-deficient rabbits injected with a serum-sensitive strain of E. coli developed infective endocarditis (P less than 0.005). No correlation was found between the presence of K1 antigen and the incidence of experimental E. coli endocarditis. Thus, the ability of strains of E. coli to establish persisting endocardial infection in rabbits appears to be directly associated with resistance to the complement-mediated serum bactericidal system. These findings may explain in part the rarity of gram-negative bacillary endocarditis in patients; they also indicate that in certain special circumstances the serum bactericidal system can play a decisive role in host defense.     

Right-sided infective endocarditis combined with mitral involvement in a patient with ventricular septal defect

An autopsy case of right-sided infective endocarditis combined with mitral valvular involvement in a 20-year-old male Japanese with ventricular septal defect (VSD) was reported. The vegetations were found on the endocardium bordering VSD, tricuspid valve, mural endocardium of the right ventricular outflow tract, and even the pulmonic valve, resulting in forming infective aneurysm of the pulmonary trunk. Streptococcus was morphologically identified in the vegetations obtained at autopsy. On the other hand, smaller vegetations were also noted on the mitral valve. The mechanisms of the mitral extending were discussed when right-sided infective endocarditis associated with VSD preceded that on the mitral valve. The authors think that mitral regurgitation in relation to VSD and right to left shunt through VSD which occur even temporarily may be the most important mechanism responsible for the mitral valvular involvement. Several differences between right-sided and left-sided infective endocarditis were also reviewed.     

Right-sided infective endocarditis as a consequence of flow-directed pulmonary-artery catheterization. A clinicopathological study of 55 autopsied patients

We studied 142 consecutively autopsied patients prospectively to determine the frequency and clinical importance of right-sided endocardial lesions in patients who had undergone flow-directed pulmonary-artery catheterization within one month of death. Of the 55 catheterized patients, 29 (53 per cent) had one or more right-sided endocardial lesions: 12 (22 per cent) had subendocardial hemorrhage, 11 (20 per cent) sterile thrombus, 2 (4 per cent) hemorrhage and thrombus, and 4 (7 per cent) infective endocarditis. Of 41 lesions seen in the 29 patients, 23 (56 per cent) were located on the pulmonic valve, 6 (15 per cent) on the tricuspid valve, 6 (15 per cent) in the right atrium, 4 (10 per cent) in the right ventricle, and 2 (5 per cent) in the main pulmonary artery. All four patients with infective endocarditis had had positive antemortem blood cultures while the catheter was in place, but in only one had the diagnosis of endocarditis been suspected clinically. The unusual locations of the infected vegetations (on the pulmonic valve in three and in the right atrium in one) and the similar location of the uninfected lesions suggest that the infective endocarditis was a consequence of catheter-induced endocardial damage with concurrent or subsequent bacteremia. Among the 87 non-catheterized patients, there were two subendocardial hemorrhages and one resolving right atrial thrombus. We conclude that endocardial damage from flow-directed pulmonary-artery catheterization is common and that right-sided infective endocarditis should be suspected in bacteremic catheterized patients.     

RIGHT-SIDED INFECTIVE ENDOCARDITIS COMBINED WITH MITRAL INVOLVEMENT IN A PATIENT WITH VENTRICULAR SEPTAL DEFECT

An autopsy case of right-sided infective endocarditis combined with mitral valvular involvement in a 20-year-old male Japanese with ventricular septal defect (VSD) was reported. The vegetations were found on the endocardium bordering VSD, tricuspid valve, mural endocardium of the right ventricular outflow tract, and even the pulmonic valve, resulting in forming infective aneurysm of the pulmonary trunk. Streptococcus was morphologically identified in the vegetations obtained at autopsy. On the other hand, smaller vegetations were also noted on the mitral valve. The mechanisms of the mitral extending were discussed when right-sided infective endocarditis associated with VSD preceded that on the mitral valve. The authors think that mitral regurgitation in relation to VSD and right to left shunt through VSD which occur even temporarily may be the most important mechanism responsible for the mitral valvular involvement. Several differences between right-sided and left-sided infective endocarditis were also reviewed. ACTA PATHOL. JPN. 35 : 459–471, 1985