title: Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model.
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 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.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. kernodds@ctrvax.vanderbilt.edu
authors.forename: D S
authors.initials: DS
authors.lastname: Kernodle
authors.forename: R K
authors.initials: RK
authors.lastname: Voladri
authors.forename: B E
authors.initials: BE
authors.lastname: Menzies
authors.forename: C C
authors.initials: CC
authors.lastname: Hager
authors.forename: K M
authors.initials: KM
authors.lastname: Edwards
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D004195:Disease Models, Animal; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D051379:Mice; D016372:RNA, Antisense / Q000096:biosynthesis* / Q000235:genetics; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D013203:Staphylococcal Infections / Q000209:etiology* / Q000401:mortality; D013211:Staphylococcus aureus / Q000235:genetics / Q000472:pathogenicity*; D016019:Survival Analysis
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; C021091:staphylococcal alpha-toxin