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Like GAS, colonies of GBS
produce a zone of
surrounding ß-hemolysis when grown on blood agar media. As
is the case
with streptolysin S of GAS, the ß-hemolysin of GBS is an
oxygen-stable, non-immunogenic, pore-forming cytolysin that has yet to
be fully purified.
GBS ß-hemolysin activity is stabilized by albumin, starch or
Tween
80 and inhibited by phospholipids such as dipalmitoyl
phosphatidylcholine
(DPPC). The chromosomal location of genes (cyl) encoding
GBS
ß-hemolysin/cytolysin activity was first discovered by Barbara
Spellerberg
and colleagues in Germany by analysis of nonhemolytic mutants generated
using
a novel transposition vector and independently by our group by mapping
the
location of a GBS DNA fragment that conferred ß-hemolysis to Escherichia
coli.
Targeted mutagenesis and
expression cloning experiments demonstrated
that the cylE ORF, encoding a predicted 78 kDa protein without GeneBank
homologies, was by itself necessary for GBS ß-hemolytic/cytolytic
activity and sufficient to confer b-hemolysis when expressed in E.
coli . The putative GBS ß-hemolysin/cytolysin
(ß-h/c) CylE
appears anomously located amid a fatty acid biosynthesis operon.
The ß-h/c phenotype is also curiously linked to GBS production of
an orange carotenoid pigment: nonhemolytic (NH) mutants are
nonpigmented
and hyperhemolytic (HH) mutants are hyperpigmented.
In vitro studies using isogenic
GBS mutants with a nonhemolytic (NH) or
hyperhemolytic (HH) phenotype have shed light on how the ß-h/c
toxin may contribute to disease pathogenesis. GBS ß-h/c
production is correlated with cytolytic injury to lung epithelial
cells, lung endothelial cells, brain endothelial cells and macrophages.
Injured cells showed surface bleb formation, dramatic loss of
cytoplasmic density, splitting of the cytoplasmic and nuclear
membranes, dilated organelles, and clumping of nuclear chromatin, all
consistent with a pore-forming mechanism of action. Our
collaborators Axel Ring and Jerry Shenep found GBS ß-h/c
stimulates iNOS transcription and NO production in macrophages, while
Philippe Henneke and Doug Golenbock showed the ß-h/c can
trigger macrophage apoptosis through a MyD88-independent pathway.
At subcytolytic doses, we found ß-h/c promotes GBS invasion of
human lung epithelial cells and triggers release of the neutrophil
chemoattractant interleukin-8 (IL-8). The ß-h/c is also the
key factor activating brain microvascular endothelial cell genes (IL-8,
GROa, ICAM-1, GM-CSF) implicated in the neutrophilic inflammatory
response of GBS meningitis. The GBS ß-h/c is cytolytic to
macrophages and also promotes their apoptosis, allowing the bacterium
to resist phagocytic clearance. Many cytolytic and proinflammatory
properties of the ß-h/c can be blocked by DPPC, the major
component of pulmonary surfactant. Lack of DPPC inhibition of
ß-h/c toxicity may contribute to the increased incidence and
severity of GBS pneumonia and sepsis in premature, surfactant-deficient
neonates.
GBS ß-h/c also
contributes to virulence in animal studies. Challenge of rabbits with
isogenic GBS mutants by collaborators Axel Ring and Jerry Shenep showed
that ß-h/c production was associated with significantly higher
mortality and evidence of liver necrosis with hepatocyte
apoptosis. Our own studies have established a strong correlation
between GBS ß-h/c production and sepsis and pneumonia in adult
mice, as well as pneumonia and bactermia in a newborn rabbits. Thus it
appears that GBS ß-h/c is a pluripotent
virulence factor that contributes to disease pathogenesis by
cytotoxicity and inflammatory activation.
Current efforts are aimed at
purification and characterization of the
GBS ß-h/c protein, understanding ß-h/c activation of
innate immune and apoptotic pathways, and exploration of the effects of
ß-h/c neutralization using in vitro and in vivo models of GBS
disease pathogenesis.
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