No abstract

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.

Background: The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins.

Rationale: Although interferon-␥ (IFN-␥) assays are promising alternatives to the tuberculin skin test (TST), their serial testing performance is unknown. Objective: To compare TST and IFN-␥ conversions and reversions in healthcare workers. Methods: We prospectively followed-up 216 medical and nursing students in India who underwent baseline and repeat testing (after 18 mo) with TST and QuantiFERON-TB Gold In-Tube (QFT). TST conversions were defined as reactions greater than or equal to 10 mm, with increments of 6 or 10 mm over baseline. QFT conversions were defined as baseline IFN-␥ less than 0.35 and follow-up IFN-␥ greater than or equal to 0.35 or 0.70 IU/ml. QFT reversions were defined as baseline IFN-␥ greater than or equal to 0.35 and followup IFN-␥ less than 0.35 IU/ml. Results: Of the 216 participants, 48 (22%) were TST-positive, and 38 (18%) were QFT-positive at baseline. Among 147 participants with concordant baseline negative results, TST conversions occurred in 14 (9.5%; 95% confidence interval [CI] ϭ 5.3-15.5) using the 6 mm increment definition, and 6 (4.1%; 95% CI ϭ 1.5-8.7) using the 10 mm increment definition. QFT conversions occurred in 17/147 participants (11.6%; 95% CI ϭ 6.9-17.9) using the definition of IFN-␥ greater than or equal to 0.35 IU/ml, and 11/147 participants (7.5%; 95% CI ϭ 3.8-13.0) using IFN-␥ greater than or equal to 0.70 IU/ml. Agreement between TST (10 mm increment) and QFT conversions (у 0.70 IU/ml) was 96% ( ϭ 0.70). QFT reversions occurred in 2/28 participants (7%) with baseline concordant positive results, as compared with 7/10 participants (70%) with baseline discordant results (p Ͻ 0.001). Conclusions: IFN-␥ assay shows promise for serial testing, but repeat results need to be interpreted carefully. To meaningfully interpret serial results, the optimal thresholds to distinguish new infections from nonspecific variations must be determined.

An estimated one-third of the world's population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. tuberculosis disrupted in the mce1 operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor ␣, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. The mce1 mutant strain complemented with the mce1 genes stimulated tumor necrosis factor ␣ and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that the mce1 operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. The mce1 operon of M. tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host. Approximately 60% of people who become infected with Mycobacterium tuberculosis develop asymptomatic latent infection (1). This reservoir of latently infected individuals has a 2-23% lifetime risk of developing active disease, referred to as reactivation tuberculosis (1). How M. tuberculosis establishes and maintains latent infection in an animal host is poorly understood. Several candidate M. tuberculosis genes have been recently reported to be important for persistence in the mouse model of tuberculosis. They include the isocitrate lyase gene (icl), mycolic acid cyclopropane synthase gene (pca), and a two-component response regulator called mprA (2)(3)(4). In each case, the disruption of the gene led to attenuation of the mutant strains in the mouse model of infection, whereas their in vitro growth kinetics remained similar to that of the respective wild-type strain (2-4).We reported previously the identification of a M. tuberculosis gene mce1A (Rv0169, Sanger Centre genome sequence designation) that conferred on a nonpathogenic Escherichia coli strain an ability to enter nonphagocytic cells (5). The encoded product facilitated uptake of synthetic microspheres into nonphagocytic cells, and an active domain of the protein was recently shown to cause cytoskeletal rearrangement in HeLa cells that was both microfilament-and microtubule-dependent (6, 7). The gene mce1A is located in a putative operon called mce1 containing 12 genes in the M. tuberculosis H37Rv genome (8) (Fig. 1A). The genome of M. tuberculosi...

TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.

SummaryPorins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We puri®ed a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the puri®ed porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were speci®cally detected by a polyclonal antiserum that was raised to puri®ed MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal signi®cant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.

In this study, the currently known typing methods forMycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.