Dendrimer-enabled transformation of Chlamydia trachomatis.
Lack of a system for genetic manipulation of Chlamydia trachomatis has been a key challenge to advancing understanding the molecular genetic basis of virulence for this bacterial pathogen. We developed a non-viral, dendrimer-enabled system for transformation of this organism and used it to characterize the effects of inserting the common 7.5 kbp chlamydial plasmid into strain L2(25667R), a C. trachomatis isolate lacking it. The plasmid was cloned in pUC19 and the clone complexed to polyamidoamine dendrimers, producing ∼83 nm spherical particles. Nearly confluent McCoy cell cultures were infected with L2(25667R) and reference strain L2(434). At 16 h post-infection, medium was replaced with dendrimer-plasmid complexes in medium lacking additives (L2(25667R)) or with additive-free medium alone (L2(434)). Three h later complexes/buffer were removed, and medium was replaced; cultures were harvested at various times post-transformation for analyses. Real time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. A previous report indicated that one or more plasmid-encoded product govern(s) transcription of the glycogen synthase gene (glgA) in standard strains. In L2(25667R) the gene is not expressed, but transformants of that strain given the cloned chlamydial plasmid increase glgA expression, as does L2(434). The cloned plasmid is retained, replicated, and expressed in transformants over at least 5 passages, and GFP is expressed when transformed into growing L2(25667R). This transformation system will allow study of chlamydial gene function in pathogenesis.
Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21235, USA.
Rangaramanujam M
RM
Kannan
Hérve C
HC
Gérard
Manoj K
MK
Mishra
Guangzhao
G
Mao
Sunxi
S
Wang
Mirabela
M
Hali
Judith A
JA
Whittum-Hudson
Alan P
AP
Hudson
D002692:Chlamydia trachomatis / Q000235:genetics*; D050091:Dendrimers; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006006:Glycogen Synthase / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics*; D014774:Virulence / Q000235:genetics
D050091:Dendrimers; D049452:Green Fluorescent Proteins; D006006:Glycogen Synthase
Chlamydia; Genetics; Pathogenesis; Plasmids; Transcription; Transformation