title: Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.
abstract: Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.
authors.affiliation: AIDS Virus Research Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham 2131, Johannesburg, South Africa. leonardd@nicd.ac.za
authors.forename: Leonard H
authors.initials: LH
authors.lastname: Damelin
authors.forename: Demetra
authors.initials: D
authors.lastname: Mavri-Damelin
authors.forename: Todd R
authors.initials: TR
authors.lastname: Klaenhammer
authors.forename: Caroline T
authors.initials: CT
authors.lastname: Tiemessen
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D019742:Chemokines, CC / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D006801:Humans; D007778:Lactobacillus / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D019742:Chemokines, CC; D011994:Recombinant Proteins