title: Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor.
abstract: Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of many infectious disease pathogens. Mycobacterial IdeR is an iron-dependent repressor that shows 80% identity in the functional domains with its corynebacterial homologue, DtxR (diphtheria toxin repressor). We have transformed Mycobacterium tuberculosis with a vector expressing an iron-independent, positive dominant, corynebacterial dtxR hyperrepressor, DtxR(E175K). Western blots of whole-cell lysates of M. tuberculosis expressing the dtxR(E175K) gene revealed the stable expression of the mutant protein in mycobacteria. BALB/c mice were infected by tail vein injection with 2 x 10(5) organisms of wild type or M. tuberculosis transformed with the dtxR mutant. At 16 weeks, there was a 1.2 log reduction in bacterial survivors in both spleen (P = 0.0002) and lungs (P = 0.006) with M. tuberculosis DtxR(E175K). A phenotypic difference in colonial morphology between the two strains also was noted. A computerized search of the M. tuberculosis genome for the palindromic consensus sequence to which DtxR and IdeR bind revealed six putative "iron boxes" within 200 bp of an ORF. Using a gel-shift assay we showed that purified DtxR binds to the operator region of five of these boxes. Attenuation of M. tuberculosis can be achieved by the insertion of a plasmid containing a constitutively active, iron-insensitive repressor, DtxR(E175K), which is a homologue of IdeR. Our results strongly suggest that IdeR controls genes essential for virulence in M. tuberculosis.
authors.affiliation: Department of Medicine, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Y C
authors.initials: YC
authors.lastname: Manabe
authors.forename: B J
authors.initials: BJ
authors.lastname: Saviola
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
authors.forename: W R
authors.initials: WR
authors.lastname: Bishai
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics*; D007501:Iron / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000472:pathogenicity*; D012097:Repressor Proteins / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014774:Virulence / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D012097:Repressor Proteins; D007501:Iron