EXPRESSION OF AN ANTISENSE HLA FRAGMENT IN STAPHYLOCOCCUS-AUREUS REDUCES ALPHA-TOXIN PRODUCTION IN-VITRO AND ATTENUATES LETHAL ACTIVITY IN A MURINE MODEL

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.