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Review
. 2016 Mar 30;80(2):411-27.
doi: 10.1128/MMBR.00071-15. Print 2016 Jun.

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

Robert J Bastidas  1 , Raphael H Valdivia  2
Affiliations
Review

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

Robert J Bastidas et al. Microbiol Mol Biol Rev. .

Abstract

Chlamydia species infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle of Chlamydia and restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome of Chlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools for Chlamydia and the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen.

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Figures

FIG 1
FIG 1
C. trachomatis LGV L2 expression vectors encoding a β-lactamase resistance marker for use in LGV L2 strains. (A) The p2TK2-SW2 plasmids (20) are derivatives of the pGFP-SW2 plasmid (19). pSW2 is a C. trachomatis LGV L2 plasmid isolated from the Swedish SW2 strain, which contains a 377-bp deletion in CDS1 (148). The p2TK2-SW2 vector features a versatile multiple-cloning site for ectopic gene expression under the control of native promoters. p2TK2-SW2 plasmids expressing rsgfp, mCherry, and cfp fluorophores from the incD promoter are ideal for generating fluorescently labeled bacteria. Fluorophore-encoding genes can be substituted with a gene of interest for expression under the control of the incD promoter. (B) pBOMB4 vectors (21) are derived from an intact C. trachomatis LGV L2 (434/Bu) plasmid. These vectors include a multiple-cloning site in addition to fluorescent markers to confirm the presence of recombinant plasmids in transformed bacteria. pBOMB4 (GenBank accession no. KF790906) and pBOMB4-MCI (GenBank accession no. KF790907) are ideal for expressing proteins from native promoters, and pBOMB4R (GenBank accession no. KF790908) and pBOMB4R-MCI (GenBank accession no. KF790909) promote constitutive protein expression from the rpoB promoter. (C) Both pBOMB4-Tet-mCherry (GenBank accession no. KF790910) (21) and pASK-GFP/mKate2-L2 (26) are derived from an intact C. trachomatis LGV L2 (434/Bu) plasmid. Protein expression is controlled by the inducible tetA promoter in both plasmids. pBOMB4-Tet-mCherry features a multiple-cloning site and encodes GFP as a fluorescent marker. pASK-GFP/mKate2-L2 encodes mKate2 as a far-red fluorescent marker for transformed strains. (D) Multiple-cloning sites in each vector. All of the unique restriction sites are labeled in red. bla, β-lactamase-encoding gene; MCS, multiple-cloning site; pSW2, plasmid from C. trachomatis LGV L2 strain SW2; pL2, plasmid from C. trachomatis LGV L2 (434/Bu) strain. (The vector maps in panel A are adapted from reference with permission, the vector maps in panel B and the pBOMB4-Tet-mCherry map in panel C are adapted from reference with permission, and the pASK-GFP/mKate2-L2 map in panel C is adapted from reference with permission.)
FIG 2
FIG 2
C. trachomatis expression vectors for use in non-LGV L2 strains. Plasmid pGFPBSD/Z::SW2 (27) is a derivative of pGFP:SW2 (19) in which the chloramphenicol acetyltransferase gene (cat) has been replaced by a blasticidin S deaminase gene (bsd) and the β-lactamase-encoding gene (bla) has been replaced by the Shble (zeocin resistance cassette) gene. pGFP-CAT::SW2 (28) is another derivative of pGFP:SW2, in which the β-lactamase-encoding gene (bla) has been removed. pGFPBSD/Z::SW2- and pGFP-CAT::SW2-transformed cells can be selected by using blasticidin and chloramphenicol, respectively. (The pGFPBSD/Z:SW2 map is adapted from reference with permission, and the pGFP-CAT::SW2 map is adapted from reference with permission.)
FIG 3
FIG 3
TargeTron vectors adapted for targeted mutagenesis in C. trachomatis. The suicide plasmid pDFTT3-bla features a group II intron carrying a bla marker that is targeted for integration into the incA locus. Intron RNA expression is driven by the CTL0655 promoter from C. trachomatis LGV L2/434/Bu. pDFTT3-aadA is a reengineered version of pDFTT3-bla in which the bla marker has been replaced with the spectinomycin resistance marker aadA. (The pDFTT3-bla map is adapted from reference with permission, and the pDFTT3-aadA map is based on data from reference .)
FIG 4
FIG 4
Strategies for genetic analyses of C. trachomatis. (A) Reverse genetic approach for selecting mutant strains harboring mutations in a gene of interest. Pools of ∼10 organisms are generated and arrayed in 96-well plates. A target of choice, such as an ORF, operon, or promoter region, is amplified from each pool, and mutated targets are identified by CEL1 digestion (TILLING). The gel image depicts a representative CEL1 digest. Sanger sequencing is then used to determine the genotypes of mutant targets in the positive pools. Individual strains carrying the mutant target of interest are isolated from each pool by a standard plaque assay. (B) Strategy for forward genetic analysis of Chlamydia. A rifampin-resistant C. trachomatis strain is mutagenized by chemical mutagenesis. Individual mutant strains are isolated by a standard plaque assay. Mutant strains are selected from phenotypic screens of plaque-purified strains, and their genomes are sequenced to identify genetic lesions. To establish linkage between a gene lesion and a phenotype, recombinant strains are selected in the presence of rifampin and spectinomycin after coinfection of host cells with a wild-type strain (Specr) and a mutant (Rifr) strain. TILLING can be utilized to follow the segregation of mutant alleles in recombinants displaying the phenotype. (C) A library of 934 plaque-purified C. trachomatis (LGV L2 434/Bu) mutants has been generated in which all single-nucleotide substitutions have been mapped by whole-genome sequencing. This collection can be utilized for phenotypic screens (forward genetic approach) or to isolate mutant strains harboring a mutant allele of interest (reverse genetic approach). Linkage between a mutant allele and a phenotype of interest can be determined as described for panel B.
FIG 5
FIG 5
C. trachomatis LGV L2 434/Bu alleles harboring nonsense single-nucleotide substitutions.
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