Attenuated strains of mycobacteria can be exploited to determine genes essential for their pathogenesis and persistence. To this goal, we sequenced the genome of H37Ra, an attenuated variant of Mycobacterium tuberculosis H37Rv strain. Comparison with H37Rv revealed three unique coding region polymorphisms. One polymorphism was located in the DNA-binding domain of the transcriptional regulator PhoP, causing the protein's diminished DNA-binding capacity. Temporal gene expression profiles showed that several genes with reduced expression in H37Ra were also repressed in an H37Rv phoP knockout strain. At later time points, genes of the dormancy regulon, typically expressed in a state of nonreplicating persistence, were upregulated in H37Ra. Complementation of H37Ra with H37Rv phoP partially restored its persistence in a murine macrophage infection model. Our approach demonstrates the feasibility of identifying minute but distinct differences between isogenic strains and illustrates the consequences of single point mutations on the survival stratagem of M. tuberculosis.
Bibliographical noteFunding Information:
This work received financial support from the European Union Framework Program 6 Project “Design and Testing of Vaccine Candidates Against Tuberculosis (TBVAC),” the Bundesministerium für Bildung und Forschung Kompetenz Netzwerk “PathoGenoMikPlus,” the National Genome Research Network 2 (Germany) to S.H.E.K., and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GM03-002) to S.-N.C. We thank Carlos Martin for providing the PhoP complementation plasmid and for discussing results and Stewart T. Cole for providing the H37Rv strain. Thanks also to Mary Louise Grossman for editorial help and Marc Jacobsen, Vladimir Yeremeev, and Arunava Dasgupta for helpful discussions.
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