| |
|
|
| Home | Research | People | Publications | Protocols | Links | Streptococci | Contact | Directions | Calendar | Photos |
|
The principal factor
responsible for
ß-hemolysis in GAS is streptolysin S (SLS), an oxygen-stable,
non-immunogenic, broad-spectrum cytolysin that has yet to be fully
purified. Insertion of SLS into the RBC membrane results in
transmembrane pore formation and osmotic cell lysis. Our research
has applied a molecular approach to discover the genetic basis of SLS
production and the role of this potent exotoxin in disease
pathogenesis. These studies were the product of collaboration
with the laboratories of Joyce DeAzavedo at Mt.
Sinai Hospital in Toronto and Bernie Beall at the Centers for Disease
Control in Atlanta. A locus of 9 contiguous ORFs associated with
SLS production
was
identified by analyzing random transposon mutants of GAS exhibiting a
nonhemolytic phenotype. This locus, conserved among GAS of
various emm genotypes, is named "sag" for
"streptolysin-associated genes" (Figure
1).
 The sag locus has many
features characteristic of a
bacteriocin biosynthetic operon. The first gene, sagA
encodes a 53 aa candidate prepropeptide (Figure 2). Within SagA
is a typical Gly-Gly cleavage motif separating an N-terminal 23 aa
leader from a 30 aa propeptide matching the calculated size of mature
SLS (2.9 kD). The propeptide is highly enriched in amino acids
(Ser, Thr, Gly, Cys) that are the precursors for
post-translational modification and thioether bond formation in other
cyclical
bacteriocin toxins. The sagG-sagI genes have
strong
homology to ATP-binding cassette (ABC) transporters commonly required
for
the export of bacteriocins peptides. The SagB and SagE predicted
proteins
share very weak homology to a bacteriocin modifying enzyme and immunity
protein,
respectively; the other sag gene products have no significant
Genbank
homologies. RT-PCR analysis confirms an operon structure, as the
sagB-sag I genes utilize the same promoter as sag
A.
As in other bacteriocin operons, a “leaky” terminator situated between
sagA and sag B acts as a regulatory mechanism yielding an
abundance
of structural gene transcript ( sag A alone) and smaller amounts
of
mRNA for downstream genes involved in modification, processing and
export
of the mature toxin.
 Plasmid integrational
mutagenesis verified the transposon
mutant phenotypes and defined the functional boundaries of the sag
operon; targeted integrations in each gene yielded nonhemolytic GAS,
while mutations upstream of the sag promoter or downstream of
sagI did not affect SLS production. Cloning of the entire
9-gene sag locus in nonhemolytic Lactococcus lactis
resulted in robust and stable ß-hemolytic transformants.
These experiments demonstrated the intact sag locus
is both necessary and sufficient for SLS production (Figure
3).
 Homologues of the GAS sag
operon for SLS biosynthesis have
recently been identified in invasive human isolates of
ß-hemolytic group C and
G streptococci and the fish pathogen S. iniae. The contribution
of
SLS to the pathogenesis of streptococcal necrotizing soft tissue
infection has been examined in the murine model of necrotizing
fasciitis. In this model, wild-type bacteria elicit a ulcer with
bacterial proliferation, neutrophilic inflammation, and histopathologic
evidence of vascular injury and tissue necrosis. In contrast to
the parent strains, isogenic SLS-negative sag gene mutants do
not develop ulcers, and biopsy of the inoculation site demonstrates
bacterial clearance and minimal degrees of inflammation or tissue
injury (Figure 4). In vitro studies suggest that SLS can
contribute to pathogenesis both by direct cytotoxicity and by
inhibiting
neutrophil phagocytosis. The latter may help explain the paradox
of
decreased bacterial clearance despite the intense neutrophil influx
seen
with the wild-type bacteria.
 Our ongoing research takes advantage of the unique genetic information and specific bacteriologic reagents we have generated to further study the basic biology and pathogenic role of the SLS toxin. Efforts include studies to (1) purify the toxin and understand its biosynthetic pathway, (2) determine the specific SagA amino acid residues critical for its cytolytic action, (3) characterize SLS antiphagocytic, proinflammatory and antibacterial properties, (4) assess the contribution of the toxin to disease pathogenesis in vivo, and (5) determine the potential benefits of SLS neutralization in the treatment of invasive infection. |
|
Home | Research Overview | Back to Top |