ABSTRACT
BACKGROUND: Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis-infected guinea pigs form caseating granulomas, which may simulate human disease more closely. METHODS: We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model. RESULTS: M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P<.0003). CONCLUSIONS: We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.
Subject(s)
Bacterial Proteins/genetics , Lung/microbiology , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/pathogenicity , Tuberculosis, Pulmonary/microbiology , Animals , Bacterial Proteins/metabolism , DNA Transposable Elements , Disease Models, Animal , Female , Guinea Pigs , Humans , Lung/pathology , Mice , Mice, Inbred BALB C , Mutagenesis, Insertional , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oligonucleotide Array Sequence Analysis , Species Specificity , Tuberculosis, Pulmonary/pathologyABSTRACT
Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The -10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.
