Group B Streptococcal b-Hemolysin Induces Nitric Oxide Production (original) (raw)
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Group B Streptococcal �-Hemolysin Induces Nitric Oxide Production in Murine Macrophages
J Infec Dis, 2000
Group B streptococcus (GBS) is the leading cause of sepsis in neonates. Nitric oxide (NO) release plays a role in the hypotension that characterizes septic shock. To examine the role of the GBS b-hemolysin in NO production, the murine macrophage line RAW 264.7 was exposed to a wild-type (WT) GBS isolate and to hyperhemolytic (HH) and nonhemolytic (NH) transposon mutants derived from that isolate. After activation of macrophages by the WT strain, the HH mutant, or cell-free extracts of b-hemolysin, nitrite release into the supernatant increased 110-fold and inducible NO synthase (iNOS) levels in cell lysates increased up to 10-fold compared with treatment with the NH mutant or extracts from that mutant. Hemolysin-induced NO production was dependent on protein tyrosine kinases and NF-kB, but not on extracellular signal-related kinase-1/2-mitogen-activated kinases or protein kinase A. These results indicate that GBS b-hemolysin induces murine macrophage iNOS via intracellular pathways similar to those that mediate lipopolysaccharide-induced iNOS activation. Group B streptococcus (GBS; Streptococcus agalactiae) is the leading cause of pneumonia, meningitis, and septic shock in newborns, particularly those born prematurely [1]. All clinical isolates are encapsulated, and 98%-99% are b-hemolytic [2]. The GBS capsular polysaccharide contributes to virulence by virtue of its antiphagocytic property [3]. The GBS b-hemolysin, a potent membrane cytotoxin that injures lung epithelial [4] and endothelial [5] and brain endothelial [6] cells, may contribute to the pathogenesis of neonatal pneumonia and meningitis. Crude b-hemolysin preparations from GBS cultures induce cardiotoxicity and hypotension after intravenous administration to rabbits or rats, suggesting a role in septic shock [7]; however, the molecular mechanisms by which the GBS b-hemolysin may contribute to the pathogenesis of septic shock have not been delineated. The hypotension of septic shock is thought to be due in part to an excess production of nitric oxide (NO), as elevated NO levels are found in septic patients [8, 9]. Three isoforms of NO synthases (NOS) are present in mammals: the high-output in
Group B Streptococcal β‐Hemolysin Induces Nitric Oxide Production in Murine Macrophages
The Journal of Infectious Diseases, 2000
Group B streptococcus (GBS) is the leading cause of sepsis in neonates. Nitric oxide (NO) release plays a role in the hypotension that characterizes septic shock. To examine the role of the GBS b-hemolysin in NO production, the murine macrophage line RAW 264.7 was exposed to a wild-type (WT) GBS isolate and to hyperhemolytic (HH) and nonhemolytic (NH) transposon mutants derived from that isolate. After activation of macrophages by the WT strain, the HH mutant, or cell-free extracts of b-hemolysin, nitrite release into the supernatant increased 110-fold and inducible NO synthase (iNOS) levels in cell lysates increased up to 10-fold compared with treatment with the NH mutant or extracts from that mutant. Hemolysin-induced NO production was dependent on protein tyrosine kinases and NF-kB, but not on extracellular signal-related kinase-1/2-mitogen-activated kinases or protein kinase A. These results indicate that GBS b-hemolysin induces murine macrophage iNOS via intracellular pathways similar to those that mediate lipopolysaccharide-induced iNOS activation. Group B streptococcus (GBS; Streptococcus agalactiae) is the leading cause of pneumonia, meningitis, and septic shock in newborns, particularly those born prematurely [1]. All clinical isolates are encapsulated, and 98%-99% are b-hemolytic [2]. The GBS capsular polysaccharide contributes to virulence by virtue of its antiphagocytic property [3]. The GBS b-hemolysin, a potent membrane cytotoxin that injures lung epithelial [4] and endothelial [5] and brain endothelial [6] cells, may contribute to the pathogenesis of neonatal pneumonia and meningitis. Crude b-hemolysin preparations from GBS cultures induce cardiotoxicity and hypotension after intravenous administration to rabbits or rats, suggesting a role in septic shock [7]; however, the molecular mechanisms by which the GBS b-hemolysin may contribute to the pathogenesis of septic shock have not been delineated. The hypotension of septic shock is thought to be due in part to an excess production of nitric oxide (NO), as elevated NO levels are found in septic patients [8, 9]. Three isoforms of NO synthases (NOS) are present in mammals: the high-output in
The Journal of Infectious Diseases, 2002
Group B streptococcus (GBS) is the leading cause of sepsis in neonates. Nitric oxide (NO) release plays a role in the hypotension that characterizes septic shock. It has been shown that GBS b-hemolysin/cytolysin (b-h/c) stimulates the transcription of inducible NO synthase (iNOS) in murine macrophages via intracellular pathways similar to those that mediate lipopolysaccharide-induced iNOS activation. Here, it is demonstrated that the GBS cell wall and b-h/c act synergistically to induce iNOS in interferon (IFN)-a-primed RAW 264.7 murine macrophages. In nonprimed macrophages, combined activation by the GBS cell wall plus bh/c is necessary to induce an NO response, which indicates that both virulence factors cooperate to substitute for the priming signal typically provided by IFN-a. Group B streptococcus (GBS) is the leading cause of pneumonia, meningitis, and septic shock in newborns, particularly those born prematurely. All clinical isolates are encapsulated, and 98%-99% are b-hemolytic [1]. GBS b-hemolytic activity is mediated by a pore-forming cytotoxin, the genetic basis of which has recently been elucidated [2]. In vitro, the GBS b-hemolysin/cytolysin (b-h/c) is associated with injury to lung epithelial, lung endothelial, and brain endothelial cells and is thus speculated to contribute to GBS penetration of host cellular barriers. Very recently, we reported that b-h/c is responsible for high mortality and liver injury in a rabbit model of GBS septic shock [3]. The hypotension of septic shock is thought to be due, in part, to an excess production of nitric oxide (NO), because elevated NO levels are found in patients with sepsis. Three isoforms of NO synthases (NOS) are present in mammals, the high-output inducible NOS (iNOS) and 2 constitutive isoforms, one originally identified in neurons and the other in endothelial cells. Activated macrophages constitute a major source of NO production. The inflammatory activation of macrophages is thought to involve 2 sequential steps, priming and triggering [4]. Priming is initiated by binding of interferon (IFN)-a to its specific receptor, which results in a number of biochemical and functional alterations that render the macrophages sensitive to triggering agents such as proinflammatory cytokines, lipopolysaccharide (LPS) [4],
1998
New Zealand White rabbits were challenged with the wild-type (wt) group B streptococci (GBS) serotype III strain (COH1) and its isogenic nonhemolytic (NH) and hyperhemolytic (HH) mutants. Mortality differed significantly between rabbits infected with the HH mutant IN40 (67%), compared with rabbits infected with the wt COH1 strain (27%) and the NH strains COH1-20 and COH1:cyl EDcat (13 % and 0%, respectively; P,:05). Histopathologically, dissemi-nated septic microabscesses surrounded by necrotic foci were found exclusively in the livers of HH mutant IN40–infected animals. Serum transaminase levels were 20-fold higher in the HH-in-fected group, compared with rabbits infected with the other strains. Positive TUNEL (in situ terminal deoxynucleotide transferase–mediated dUTP nick end labeling) staining and activation of caspase-3 in hepatocytes were more frequent in HH-infected than in wt-infected animals and absent in the NH mutant COH1-20–infected group, indicating that GBS b-hemolysin...
Infection and …, 1999
Nitric oxide (NO) production by inducible NO synthase (iNOS) during inflammation is an essential element of antimicrobial immunity but can also contribute to host-induced tissue damage. Under conditions of bacterial sepsis, large amounts of NO are produced, causing hypotension, a critical pathological feature of septic shock. In sepsis caused by gram-positive organisms, the bacterial factors contributing to host NO production are poorly characterized. We show that a soluble toxin of Streptococcus pneumoniae, pneumolysin (Pln), is a key component initiating NO production from macrophages. In contrast to wild-type bacteria, a mutant of S. pneumoniae lacking Pln failed to elicit NO production from murine macrophages. Purified recombinant Pln induced NO production at low concentrations and independently of exogenous gamma interferon (IFN-␥) priming of RAW 264.7 macrophages. However, IFN-␥ was essential for Pln-induced NO production, since primary macrophages from mice lacking the IFN-␥ receptor or interferon regulatory factor 1, a transcription factor essential for iNOS expression, failed to produce NO when stimulated with Pln. In addition, Pln acts as an agonist of tumor necrosis factor alpha and interleukin 6 production in macrophages. The properties of Pln, previously identified as a pore-forming hemolysin, also include a role as a general inflammatory agonist.
Compartmentalised inducible nitric-oxide synthase activity in septic shock
Lancet, 2000
Previous experimental studies support a role for inducible nitric-oxide synthase (iNOS) in the pathogenesis of severe sepsis. The aim of the study was to characterise iNOS activity in different tissues in patients with septic shock.13 consecutive patients with septic shock caused by cellulitis were enrolled. Skin, muscle, fat, and artery samples were obtained from normal, inflamed, and putrescent areas to measure iNOS activity, and concentrations of tumour necrosis factor α (TNFα) and interleukin 1β (IL-1β). In two patients, iNOS activity was also assessed in peripheral blood mononuclear cells (PBMC) incubated with microorganisms causing the sepsis, or in macrophages isolated from suppurating peritoneal fluid incubated with IL-1β.Compared with normal and inflamed areas, iNOS activity was increased in putrescent areas for muscle (71-fold [95% CI 20–259] vs normal areas, 69-fold [19–246] vs inflammed areas; p<0·01 for each) and for fat (68-fold [23–199] and 49-fold [18–137], respectively; p<0·01), but not for skin. Compared with normal areas, putrescent areas of arteries showed increased iNOS expression (1280-fold [598–3153]; p<0·01). Compared with normal areas, TNFα and IL-1β were increased in putrescent areas of arteries (223-fold and 41-fold, respectively; p<0·01 for each). PBMCs and tissue macrophages expressed iNOS. Plasma nitrite/nitrate concentrations inversely correlated with mean arterial pressure and systemic vascular resistance.In human septic shock we found that iNOS activity is compartmentalised at the very site of infection and parallels expression of TNFα and IL-1β. PBMCs and tissue macrophages can be a cellular source for iNOS.