EXPRESSION OF AN ANTISENSE HLA FRAGMENT IN STAPHYLOCOCCUS-AUREUS REDUCES ALPHA-TOXIN PRODUCTION IN-VITRO AND ATTENUATES LETHAL ACTIVITY IN A MURINE MODEL
Ds. Kernodle et al., EXPRESSION OF AN ANTISENSE HLA FRAGMENT IN STAPHYLOCOCCUS-AUREUS REDUCES ALPHA-TOXIN PRODUCTION IN-VITRO AND ATTENUATES LETHAL ACTIVITY IN A MURINE MODEL, Infection and immunity, 65(1), 1997, pp. 179-184
Isogeneic bacterial strains that differ only in the production of a si
ngle microbial factor have been invaluable in studying the pathogenesi
s of bacterial infections, The targeted, intentional inactivation of a
gene encoding a potential virulence determinant generally requires ho
mologous recombination to replace the gene with an inactivated allele.
To determine whether the insertion and expression of a fragment of a
bacterial gene in an antisense orientation could be used as a rapid al
ternative to allelic inactivation for producing paired isogeneic isola
tes, we inverted a 600-bp fragment of the Staphylococcus aureus gene e
ncoding alpha-toxin, hla, behind its native promoter on an Escherichia
coli-S, aureus shuttle vector. A transformant of an S, aureus strain
carrying the antisense hla fragment produced antisense hla RNA and mad
e 16-fold less alpha-toxin than either its parent or an isogeneic tran
sformant containing vector DNA without kla. Also, intraperitoneal inje
ction of 1,5 x 10(9) CFU of the antisense hla-containing transformant
was significantly less lethal in a murine model than that of the paren
t (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant
without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]), We conc
lude that the expression of a fragment of hla in an antisense orientat
ion in S, aureus on a plasmid vector reduces alpha-toxin production an
d the lethal activity of the strain in a murine model, The antisense s
trategy for creating isogeneic strains of bacteria may facilitate mole
cular investigations into the pathogenesis of infection, It also may b
e useful in creating novel live-attenuated strains of bacteria for use
as vaccine candidates.