The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devR⌬) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.Mycobacterium tuberculosis is an extraordinarily successful pathogen that currently infects approximately one-third of the global population and causes 8 million new cases of tuberculosis annually (41). The tubercle bacillus functions within several hostile environments in order to survive within the human host and cause disease. For instance, the bacteria must be able to gain entry into macrophages, multiply intracellularly, survive within the lung granuloma for years, and disperse to a new host via aerosols (9). It is therefore likely that the expression of different sets of genes by M. tuberculosis at various stages of infection is crucial to its survival. Two-component regulatory systems (2CRs) are widely distributed among bacteria and plants and enable the organisms to respond to many different external stimuli (20,37,38). These systems form a large family of related proteins that consist of a membrane-bound sensor protein that activates an effector protein, generally a transcriptional regulator, by phosphorylation. 2CRs have been shown to play a crucial role in the controlled expression of virulence genes in other bacteria (11). For example, in Salmonella enterica serovar Typhimurium, the membrane-bound sensor protein PhoQ is activated by changing levels of magnesium, and this activates the PhoP response regulator. PhoP is a DNA-binding protein that binds in a sequence-specific manner to selected promoters, thereby inducing or repressing their transcription, and S. enterica serovar Typhimurium phoP mutants are highly attenuated (14, 17).The genome of M. tuberculosis contains 11 paired 2CRs, four isolated regulators, and three isolated sensors (6). Mutants in two of these, phoP and mprA are highly attenuated in a murine model (31, 43), while a prrA mutant grows more slowly in macrophages (12), indicating that these regulators control genes which are important f...

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Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.