Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

Douglas S. Kernodle, Rama K.R. Voladri, Barbara Menzies, Cynthia C. Hager, Kathryn M. Edwards

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

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 109 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.

Original languageEnglish (US)
Pages (from-to)179-184
Number of pages6
JournalInfection and Immunity
Volume65
Issue number1
StatePublished - Jan 1 1997
Externally publishedYes

Fingerprint

Staphylococcus aureus
Bacteria
Bacterial Genes
Antisense RNA
Genetic Vectors
Homologous Recombination
Gene Silencing
Intraperitoneal Injections
Bacterial Infections
Genes
Virulence
Plasmids
Vaccines
Alleles
Escherichia coli
staphylococcal alpha-toxin
In Vitro Techniques
DNA
Infection

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model. / Kernodle, Douglas S.; Voladri, Rama K.R.; Menzies, Barbara; Hager, Cynthia C.; Edwards, Kathryn M.

In: Infection and Immunity, Vol. 65, No. 1, 01.01.1997, p. 179-184.

Research output: Contribution to journalArticle

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