Characterization of the Mycobacterium tuberculosis sigma factor SigM by assessment of virulence and identification of SigM-dependent genes.

Alternate sigma factors have been implicated in the survival of mycobacteria in response to specific stresses. To characterize the role of SigM in Mycobacterium tuberculosis, a sigM deletion mutant was generated by allelic exchange in the virulent CDC1551 strain. Comparing the wild-type and Delta sigM strains by complete genomic microarray, we observed a low level of baseline expression of sigM in wild-type M. tuberculosis and no significant differences in the gene expression patterns between these two strains. Alternatively, a SigM-overexpressing M. tuberculosis strain was constructed and microarray profiling revealed SigM-dependent expression of a relatively small group of genes, which included four esat-6 homologues: esxE, esxF, esxT, and esxU. An assessment of SigM-dependent promoters from the microarray analysis revealed a putative consensus sequence for M. tuberculosis SigM of -35 GGAAC and -10 CGTCR. In vitro expression studies showed that M. tuberculosis sigM transcripts accumulate slightly in stationary phase and following heat shock. To understand the role of SigM in pathogenesis, the M. tuberculosis sigM deletion strain was compared with the isogenic wild-type strain and the complemented mutant strain for survival in murine macrophages and in the mouse model. The mutant was found to have similar abilities to survive in both the resting and activated J774A.1 macrophages. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as that of the wild-type and complemented strains in lung and spleen tissues. In time-to-death experiments in the mouse model, the Delta sigM mutant exhibited lethality times comparable to those observed for the wild-type and complemented strains. These data indicate that M. tuberculosis SigM governs the expression of a small set of genes, including four esat-6 homologues, and that the loss of sigM does not confer a detectable virulence defect in the macrophages and mouse models of infection.

Deletion of a Mycobacterium tuberculosis proteasomal ATPase homologue gene produces a slow-growing strain that persists in host tissues.

The in vivo rate of proliferation of Mycobacterium tuberculosis, the causative agent of tuberculosis, has been linked to the rate of progression and severity of disease. Here, we report that deletion of the gene MT2175 (Rv2115c), a putative mycobacterial proteasome-associated AAA-ATPase, leads to a reduction in the growth rate of M. tuberculosis in vitro and in vivo. Despite the reduced growth, the mutant persisted, with slow and gradual clearance in mouse lungs. The mutant elicited reduced levels of interferon-gamma production in the lungs and, when used as an immunizing agent, provided significant protection against challenge with a virulent strain of M. tuberculosis. Expression of the genes lat and MT3159 were highly up-regulated in the mutant. Thus, loss of MT2175 slows both in vitro and in vivo growth rates and compromises the lethality of M. tuberculosis in mice but has a minimal impact on the organism's ability to persist in host tissues.

Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements.

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.

Modeling in vivo pharmacokinetics and pharmacodynamics of moxifloxacin therapy for Mycobacterium tuberculosis infection by using a novel cartridge system.

To study the efficacy of moxifloxacin treatment for tuberculosis, we utilized a novel cartridge system to simulate in vivo pharmacokinetics. We found this system to be a robust method for modeling in vivo pharmacokinetics and present data supporting the utility of intermittent moxifloxacin treatment as a component of antituberculosis chemotherapy.

Dormancy phenotype displayed by extracellular Mycobacterium tuberculosis within artificial granulomas in mice.

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel(Mtb) is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-gamma dependent.

Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis.

The rabbit model of tuberculosis has been used historically to differentiate between Mycobacterium tuberculosis and Mycobacterium bovis based on their relative virulence in this animal host. M. tuberculosis infection in market rabbits is cleared over time, whereas infection with M. bovis results in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits to M. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of the M. tuberculosis Erdman strain were required than of M. tuberculosis H37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only with M. bovis infection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences between M. tuberculosis strains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specific M. tuberculosis genes important in human disease.

Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology.

A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon gammadelta is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.