Knock-out mice with defined major histocompatibility complex (MHC) deficiencies were infected intravenously with Mycobacterium bovis bacille Calmette Guérin (M. bovis BCG) to assess the relative impact of MHC class I- and II-dependent immune responses. Heterozygous control mice were capable of controlling growth of M. bovis BCG, although infection progressed chronically, as assessed by determination of colony-forming units. Furthermore, infected controls developed granulomatous lesions at the site of mycobacterial growth and delayed-type hypersensitivity (DTH) reactions after challenge with purified protein derivative of tuberculin. In vitro, spleen cells from heterozygous control mice produced high concentrations of interferon-gamma (IFN-gamma) after restimulation with mycobacterial antigens. In contrast, the MHC class II-deficient A beta-/- mice, which are virtually devoid of functional CD4 T cells, succumbed to M. bovis BCG infection. Furthermore, A beta-/- mice lacked DTH reactions to tuberculin and only few minute picnotic lesions were formed in livers of infected mice. Finally, spleen cells from infected A beta-/- mice failed to produce measurable IFN-gamma concentrations after restimulation in vitro with various mycobacterial antigen preparations. The capacity of beta 2-microglobulin (beta 2m)-deficient mice, which are devoid of CD8 alpha/beta T cells, to inhibit growth of M. bovis BCG was only slightly affected at low inocula, although significantly higher colony-forming units were detected in spleens. These knock-out mice developed strong DTH responses to tuberculin and their spleen cells produced high levels of IFN-gamma once reactivated by mycobacterial antigens. Furthermore, in livers of infected beta 2m-deficient mice, extravascular infiltrates developed which were more diffuse than those in infected control littermates. Remarkably, the beta 2m-deficient mice were substantially more susceptible to higher inocula of M. bovis BCG than their control littermates. Our data formally prove the essential role of MHC class II-dependent immune mechanisms in all relevant aspects of immunity to M. bovis BCG. In addition, our findings emphasize an important contribution of MHC class I-dependent immunity to effective anti-mycobacterial protection. We assume that CD4 T cells are highly effective in containing M. bovis BCG within distinct granulomatous lesions, but fail to eradicate their intracellular pathogens. It appears most likely that CD8 T cells are also required to achieve this goal.

We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome (“metabolic chokepoints”) and their similarity to known “druggable” protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as “persistence.” The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets.

The 198-amino-acid in-frame insertion in the gyrA gene of Mycobacterium xenopi is the smallest known naturally occurring active protein splicing element (intein). Comparison with other mycobacterial gyrA inteins suggests that the M. xenopi intein underwent a complex series of events including (i) removal of 222 amino acids that encompass most of the central intein domain, and (ii) addition of a linker of unrelated residues. This naturally occurring genetic rearrangement is a representative characteristic of the taxon. The deletion process removes the conserved motifs involved in homing endonuclease activity. The linker insertion represents a structural requirement, as its mutation resulted in failure to splice. The M. xenopi GyrA intein thus provides a paradigm for a minimal protein splicing element.Protein splicing elements (termed inteins) were first described in 1990 as in-frame insertions in the Saccharomyces cerevisiae VMA1 gene (9, 11). Protein splicing involves the removal of the intein from a precursor protein and the ligation of the two flanking sequences to produce a mature protein (14). Inteins are bifunctional proteins that sometimes have homing endonuclease activity, which is essential for mobility of intein genes (5,8,10,(13)(14)(15). Intein motifs involved in endonuclease activity (blocks C, D, and E) are found in the central region of the intein, whereas intein motifs involved in protein splicing are found in the terminal intein motifs (blocks A, B, F, and G) (14, 15). As of April 1, 1997, 41 putative intein sequences have been published or are available from public databases (14). Except for the Porphyra purpurea DnaB intein (150 amino acids) (16,17), most putative inteins range in size from 335 to 548 amino acids (aa) (14).Here, we report the characterization of a novel 198-aa Mycobacterium xenopi GyrA intein (Mxe GyrA intein) by sequence analysis, taxonomic significance, and splicing activity. This small intein has lost the residues required for endonuclease activity and may thus represent the minimal structural and functional information required for protein splicing. MATERIALS AND METHODSIdentification and sequence analysis of the M. xenopi intein. A collection of 29 mycobacterial species and subspecies (20) were investigated by PCR (30 cycles, with each cycle consisting of 30 s at 94°C and 30 s at 70°C) with primers H49 and H50 (7), which anneal to conserved regions in mycobacterial gyrA genes. The 991-bp amplification fragment from M. xenopi was sequenced directly from the PCR product and upon cloning into pT7T3 U13 (Pharmacia). The deducted protein sequence was aligned with other GyrA inteins (7) by using the Genetics Computer Group software and characterized by using BLAST, BLOCKS, and PredictProtein.Analysis of splicing and cleavage activities. (i) MIP constructs. Mxe gyrA inteins were expressed in the MIP (M for maltose-binding protein, I for intein, and P for paramyosin) system (21) by substituting the Mxe gyrA intein for the Pyrococcus sp. GB-D pol intein-1 which was inserted in-fram...

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