Generation of targeted Chlamydia trachomatis null mutants

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2011-04, Vol.108 (17), p.7189-7193
Main Authors: Kari, Laszlo, Goheen, Morgan M, Randall, Linnell B, Taylor, Lacey D, Carlson, John H, Whitmire, William M, Virok, Dezso, Rajaram, Krithika, Endresz, Valeria, McClarty, Grant, Nelson, David E, Caldwell, Harlan D
Format: Article
Language:English
Subjects:
anti-infective properties
Antineoplastic Agents, Alkylating - pharmacology
Biological Sciences
Chlamydia
Chlamydia Infections - enzymology
Chlamydia Infections - genetics
Chlamydia trachomatis
Chlamydia trachomatis - enzymology
Chlamydia trachomatis - genetics
disease control
ethyl methanesulfonate
Ethyl Methanesulfonate - pharmacology
genes
Genetic mutation
Genetics
Genomes
Genomics
HeLa cells
Humans
Indoles
Libraries
Medical genetics
Mutagenesis
mutants
Mutation
Pathogenesis
Pathogens
Polymerase chain reaction
sequence analysis
Sequencing
sexually transmitted diseases
starvation
tryptophan
tryptophan synthase
Tryptophan Synthase - genetics
Tryptophan Synthase - metabolism
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1102229108