Abstract
Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron’s substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA− mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.
Highlights
The Chlamydia are obligate, intracellular bacterial pathogens that infect humans as well as a wide variety of animals including economically important poultry and livestock [1]
Of paramount importance to human health is C. trachomatis, the causative agent of sexually transmitted infections and trachoma, the former of which remains the leading reportable bacterial infection both in the United States and world-wide [2,3]. These pathogens share a unique physiology in which the bacteria undergo a biphasic developmental cycle initiated when the extracellular, non-replicative form known as the elementary body (EB) binds to the surface of a susceptible eukaryotic cell [4]
C. trachomatis The base TargeTronTM vector pACD4K-C was chosen for modification for use in C. trachomatis (Figure S1A)
Summary
The Chlamydia are obligate, intracellular bacterial pathogens that infect humans as well as a wide variety of animals including economically important poultry and livestock [1]. Of paramount importance to human health is C. trachomatis, the causative agent of sexually transmitted infections and trachoma, the former of which remains the leading reportable bacterial infection both in the United States and world-wide [2,3]. These pathogens share a unique physiology in which the bacteria undergo a biphasic developmental cycle initiated when the extracellular, non-replicative form known as the elementary body (EB) binds to the surface of a susceptible eukaryotic cell [4]. After approximately 15 hours, depending upon the species, the RBs begin to convert back to EBs and are released from the cell either by cell lysis or inclusion extrusion after 40–72 hours [7]
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