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Infect. Immun., 01 1997, 179-184, Vol 65, No. 1
Copyright © 1997, American Society for Microbiology

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, RK Voladri, BE Menzies, CC Hager and KM Edwards
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. kernodds@ctrvax.vanderbilt.edu

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous 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 alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding 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 made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection 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 parent (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 conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live- attenuated strains of bacteria for use as vaccine candidates.


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J. Bacteriol. J. Virol. Eukaryot. Cell
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Copyright © 1997 by the American Society for Microbiology. All rights reserved.