Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector

Abstract

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.

Author Summary

C. trachomatis is a major human pathogen for which there is no means of genetically manipulating its DNA. It is an obligate intracellular bacterium which has a complex developmental cycle that takes place in a specialized host cell cytoplasmic vacuole known as an inclusion. We have constructed a shuttle vector based on the chlamydial plasmid and developed a new approach to select genetically modified bacteria. It uses rescue by selection of stable infectious, penicillin-resistant C. trachomatis from a pool of non-dividing, non-infectious C. trachomatis (induced by penicillin arrest of the developmental cycle). The transformed C. trachomatis is also cured of its endogenous plasmid by the selection of the transforming vector. The vector was modified to express the green fluorescent protein (GFP) in both Chlamydia and E. coli. We have genetically manipulated a plasmid-free recipient strain of C. trachomatis and shown restoration of the ability of this recipient strain to synthesize and accumulate glycogen within inclusions upon acquisition of the shuttle vector. The ability to transform and manipulate C. trachomatis using a complementation based vector is an important advance that opens up the field of Chlamydia research.