Tuberculosis is an infectious disease of enormous and increasing global importance. Currently, about one third of all humans are latently infected with its etiologic agent, Mycobacterium tuberculosis
(Mtb), and an estimated 2.5 million people die of tuberculosis annually [1
]. After infection of a mammalian host, Mtb is able to resist innate host defenses sufficiently to increase the local bacterial burden and disseminate throughout the body. With the onset of the adaptive immune response, however, the bacterial numbers are controlled in over 90% of infected individuals. Nevertheless, the host is not able to completely clear the bacterial burden, thus leading to persistence of Mtb within the lungs and other tissues of healthy individuals. These latent infections can be reactivated to generate full-blown disease, a process that is accelerated by immunocompromised states resulting from senescence, malnutrition, and co-infection with HIV, which is a major source of morbidity and mortality associated with the current HIV epidemics in many countries [2
Programmed cell death (apoptosis) plays an important role in the innate immune response against pathogens and comprises an evolutionarily conserved defense strategy that extends even into the plant world [6
]. It is therefore essential for persisting intracellular pathogens to have strong anti-apoptosis mechanisms [8
]. While a few studies have suggested that under some conditions Mtb may induce host cell apoptosis [13
], a substantial body of evidence points strongly to the expression of strong anti-apoptotic mechanisms by Mtb and other closely related virulent bacteria. Furthermore, this capacity is not found in avirulent species, suggesting a causal link between virulence and inhibition of macrophage apoptosis [17
]. This hypothesis is supported by the recent discovery that the genetic predisposition of different inbred mouse strains to mycobacterial infections is linked to the capacity of their macrophages to undergo apoptosis or necrosis upon infection, with the former response imparting a resistant and the latter a susceptible host phenotype [20
Further confirmation of the findings that Mtb inhibits host cell apoptosis is provided by a number of studies that have addressed its molecular mechanism. The importance of Mtb-induced upregulation of anti-apoptosis genes in infected macrophages for apoptosis inhibition was supported by functional data using either anti-sense oligonucleotides to knock down mcl-1 expression [19
] or A1 knock-out mice lacking the anti-apoptosis gene A1 [21
]. These results implicate the intrinsic (mitochondria-mediated) apoptosis pathway as a target for Mtb-mediated apoptosis inhibition, because mcl-1 and A1 are both members of the large family of Bcl-2–like proteins that localize prominently to mitochondria. However, this is contradicted by the finding that overexpression of Bcl-2 (another mitochondrial anti-apoptotic protein) could not rescue cells from undergoing apoptosis after infection with nonvirulent mycobacteria, thus suggesting that the extrinsic pathway (death receptor–mediated) is involved in the infection-induced apoptosis [23
]. Consistently, virulent Mtb strains could inhibit FasL-induced apoptosis in Fas-expressing cells [18
]. The same group reported very recently that lipoglycans of the Mtb cell wall stimulate the activation of NF-kB via TLR-2 and that the subsequent upregulation of cellular FLIP leads to inhibition of FasL-mediated apoptosis [24
]. Furthermore, it was suggested that Mtb stimulated the secretion of soluble TNF-R2, which led to the reduction of bioactive TNF-α in the medium and therefore less stimulation of the TNF-R1 [25
]. Altogether, it seems that virulent Mtb is able to inhibit induction of host cell apoptosis via multiple pathways, and probably encodes mechanisms to interfere with both intrinsic and extrinsic pathways for initiation of programmed cell death.
The inhibition of macrophage apoptosis by Mtb is believed to provide a number of advantages to the bacterium in its struggle to resist the host immune response. These include preservation of a favorable host cell environment during growth and persistence [26
], evasion of apoptosis-linked bactericidal effects [28
], and avoidance of efficient cytotoxic T cell priming via the detour pathway of antigen cross-presentation [15
]. This last point is of potential importance to the improvement of tuberculosis vaccines, because attenuated mycobacterial strains that induce higher levels of host cell apoptosis would be expected to stimulate more robust cellular immunity, as suggested by a recent study using recombinant M. bovis
Bacille Calmette-Guérin (BCG) expressing listeriolysin [33
]. Therefore, the identification of mycobacterial genes required for prevention of apoptosis could lead to specific strategies for designing more efficacious forms of BCG or other attenuated mycobacterial vaccine strains.
In order to clarify the role of mycobacteria in host cell apoptosis and to address its importance for bacterial virulence, we sought to identify anti-apoptosis genes via a gain-of-function genetic screen. Using this approach, we successfully identified two independent genomic regions of virulent Mtb (strain H37Rv) that mediate the inhibition of host cell apoptosis by the facultative pathogen M. kansasii. The analysis of a defined set of bacterial mutants of M. kansasii in immunocompromised (SCID) mice demonstrated a causal relationship between inhibition of apoptosis and virulence. These findings were confirmed via a loss-of-function strategy using the newly identified anti-apoptosis gene, nuoG, and demonstrated attenuation of Mtb nuoG mutants in immunocompromised and immunocompetent mice. Altogether our findings allowed, to our knowledge for the first time, the demonstration of a causal relationship between inhibition of host cell apoptosis and virulence of mycobacteria.