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. 2011 Apr 26;108(17):7189-93.
doi: 10.1073/pnas.1102229108. Epub 2011 Apr 11.

Generation of targeted Chlamydia trachomatis null mutants

Laszlo Kari  1 , Morgan M Goheen, Linnell B Randall, Lacey D Taylor, John H Carlson, William M Whitmire, Dezso Virok, Krithika Rajaram, Valeria Endresz, Grant McClarty, David E Nelson, Harlan D Caldwell
Affiliations

Generation of targeted Chlamydia trachomatis null mutants

Laszlo Kari et al. Proc Natl Acad Sci U S A. .

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effects of EMS mutagenesis on chlamydial survival and frequency of rifampicin resistance (RifR). C. trachomatis–infected McCoy cells were treated with various concentrations of EMS at 19 h postinfection. Chlamydiae were harvested 28 h after treatment and assayed for infectivity and resistance to rifampicin. (A) EMS killing curve. (B) The frequency of RifR after EMS mutagenesis. Plaque assays were performed on EMS-treated organisms in the presence or absence of rifampicin (0.1 μg/mL) to quantify resistant organisms. The frequency of RifR increased proportionately with increasing concentrations of EMS. A calculated mutagenesis frequency that would result in one mutation per chlamydial genome corresponded to 1.16 × 10−5 RifR frequency (indicated by the dashed line).
Fig. 2.
Fig. 2.
The reverse-genetic approach used to generate targeted mutations in the trpBA operon. (A) Schematic for generating and screening libraries. (B) Identification of subpopulations harboring trpBA mutations by CEL I digestion of the PCR-amplified trpBA operon followed by DNA gel electrophoretic analysis. The gel shows an example of the result of CEL I digestion of 18 subpopulations. Subpopulation in lane 7 contains a trpBA mutant, as is clearly indicated by the appearance of CEL I digestion products. (C) Confirmation and identification of the trpBA mutation by capillary sequencing. The trpBA PCR amplicon from subpopulation lane 7 was capillary sequenced. The chromatogram revealed a small secondary peak at the mutational site, confirming the presence of a SNP in trpB and identifying it as a C to T transition at nucleotide 532. (D) Summary of the 24 trpBA mutations identified in the analysis of the library of subpopulations. CEL I digestion of the 24 subpopulations harboring trpBA mutations are shown on the gels in the order of their genomic locations. The trpB and trpA ORFs are illustrated by solid yellow arrows. Synonymous (blue) and nonsynonymous (red) SNPs identified by capillary sequencing are indicated below each sample. Locations of SNPs are indicated in the trpBA operon above the gel image. Genomic scale and the region corresponding to the PCR amplicon are also shown. The nonsense mutation (black) in trpB truncates the ORF by 186 bp.
Fig. 3.
Fig. 3.
Genetic and phenotypic characterization of the CTD trpB− nonsense mutant. (A) Chromatograms corresponding to the region of the trpB nonsense mutation in the original subpopulation (V7-F10) containing the trpB nonsense mutant, the plaque-cloned trpB nonsense mutant (CTD trpB−), and the parental clone (CTD) with the wild-type trpB genotype. Asterisk indicates a stop codon. (B) Western blotting with anti-TrpA and -TrpB antibodies of HeLa cell lysates infected with CTD, CTD trpB−, and a C. trachomatis ocular strain (A2497) with a frameshift mutation in trpA. CTD expressed both the 42-kDa TrpB and 26-kDa TrpA, whereas CTD trpB− expressed TrpA but not the 42-kDa TrpB. Strain A2497 expressed TrpB but not TrpA. Anti-HSP60 mAb served as a loading control. (C) Chlamydial inclusion formation during IFN-γ–induced tryptophan starvation and supplementation of the culture medium with no tryptophan, tryptophan, or indole detected by immunofluorescent staining with anti-chlamydial antibody (green) and DAPI (blue) for DNA. (D) Recoverable IFU as measured after a similar rescue experiment (n = 3). Infectivity of all strains was reduced by 4.5–5 log10 in the absence of tryptophan compared with IFU titers after tryptophan supplementation. The infectivity of CTD was fully rescued from tryptophan starvation by indole, whereas the isogenic CTD trpB− mutant and A2497 were not rescuable by indole. Shown are means and SD.
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