Site-Specific, Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron

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.

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity

“…The validation of a protein phosphatase with broad substrate specificity further supports protein phosphorylation as a regulatory mechanism within Chlamydia. Consistent with PknD inhibitor studies that demonstrated the essentiality of protein phosphorylation for chlamydial growth, we have been unable to obtain ctl0511 insertion mutants using a group II intron mutagenesis approach (73), and Kokes et al did not obtain nonsense ctl0511 mutants using a high-content chemical mutagenesis approach (23). Future studies will focus on identifying CTL0511 inhibitors and substrates to further query the physiological role of protein phosphorylation in Chlamydia.…”

A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion

“…C. trachomatis serovar L2 was transformed with pACT-incA as previously described (14,15,28). Plasmid DNA was transformed into C. trachomatis serovar L2 (LGV 434/Bu) density gradient-purified EBs using CaCl 2 buffer (10 mM Tris [pH 7.5], 50 mM CaCl 2 ).…”

“…Recent advances, including plasmid transformation (14,25), generation of random mutants through chemical mutagenesis (26,27), and adaption of a group II intron-based approach allowing selectable site-specific gene inactivation (28), have facilitated studies aimed at identifying and characterizing chlamydial virulence factors. In the current report, we highlight the importance of IncA in mediating homotypic inclusion fusion using these recently developed genetic approaches.…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…3) for use in C. trachomatis by placing the group II intron under the control of a C. trachomatis LGV L2-specific promoter (CTL0655p) and inserting bla for selection in C. trachomatis LGV L2 biovars (33). In proof-of-principle experiments, the resulting vector was retargeted for homing into the incA locus, which encodes an inclusion membrane protein that mediates homotypic inclusion fusion (156,157).…”

“…Consequently, the use of chemical mutagenesis coupled with whole-genome sequencing and mismatch-specific endonucleases for mapping mutant alleles led to the emergence of experimental platforms to perform forward and reverse genetic screens in Chlamydiae (30)(31)(32). More recently, targeted mutagenesis was achieved using a group II intron-based gene knockout system (TargeTron) (33). These advances, although recent, promise to revolutionize our understanding of the molecular basis of Chlamydia pathogenesis.…”