Analysis of the Virulence Determinants of Group A
Streptococci
Group A Streptococci (GAS) are the cause of serious
infections such as toxic shock syndrome and necrotizing fasciitis.
The virulence factors contributing to disease are not well understood.
In order to examine the role of these, Tn916 transposon mutants
were generated. Two mutants were chosen for detailed study, one
deficient in streptolysin S(SLS) production and the other showing
expression of a capsule not present in the wild type suggesting
insertion in a regulatory element. The SLS-negative mutant was shown
to be less virulent in a mouse model of subcutaneous infection whereas
the encapsulated mutant is more virulent. The open reading frame
responsible for SLS production was cloned and sequenced and designated
sagA for streptolysin-associated gene. The estimated translational
product of 53 amino acids has 7 cysteine residues, 5 of them being
adjacent to each other. Several features of the SAG-A protein suggest
that it could be a bacteriocin or lantibiotic-like product. Using
Tn917 mutagenesis and subsequent chromosome walking steps, we have
identified a gene cluster immediately downstream of sagA that we
propose is required for the processing and transport of SAG-A/SLS.
Inactivation of each of the downstream genes by homologous recombination,
using the temperature sensitive insertional mutagenesis plasmid
pVE6007, results in a non-hemolytic phenotype. In the mouse skin
infection model, mutants with an SLS-negative phenotype, although
persisting at the site of injection after 5 days, failed to produce
necrotic lesions as seen with the wild-type organism. Re-introduction
of sagA and its promotor on a plasmid partially restored the a-hemolytic
phenotype of a sagA mutant. The ability to complement sagA in trans,
together with Northern blot analysis, suggest that the sagA message
is independently transcribed, and that sagA may be the structural
gene for SLS. We are continuing to explore the genetic basis of
SLS expression and its role in the pathogenesis of necrotic soft
tissue infection.
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