A genomic locus called "region of difference 1" (RD1) in Mycobacterium tuberculosis has been shown to contribute to the generation of host protective immunity as well as to the virulence of the bacterium. To gain insight into the molecular mechanism, we investigated the difference in the cytokine-inducing ability between H37Rv and a mutant strain deficient for RD1 (⌬RD1). We found that RD1 is implicated in the production of caspase-1-dependent cytokines, interleukin-18 (IL-18) and IL-1␤, from infected macrophages. The expression of these cytokines was similarly induced after infection with H37Rv and ⌬RD1. However, the activation of caspase-1 was observed only in H37Rv-infected macrophages. The cytokine production and caspase-1 activation were induced independently of type I interferon receptor signaling events. We also found that the activation of caspase-1 was markedly inhibited with increasing concentrations of extracellular KCl. Furthermore, the production of IL-18 and IL-1␤ and caspase-1 activation were induced independently of a P2X7 purinergic receptor, and the inability of ⌬RD1 in caspase-1 activation was compensated for by nigericin, an agent inducing the potassium ion efflux. Based on these results, we concluded that RD1 participates in caspase-1-dependent cytokine production via induction of the potassium ion efflux in infected macrophages.

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PPE37 is a member of the Mycobacterium tuberculosis proline-proline-glutamic acid (PPE) multigene family. Its expression is upregulated in bacteria that are phagocytosed by macrophages and is enhanced even more in bacteria isolated from the lungs of infected mice. This raises the possibility that PPE37 may play a role in the virulence of M. tuberculosis and led to this investigation of the function of PPE37. Recombinant bacterial strains, one expressing the M. tuberculosis PPE37 protein (Ms_ppe37) and another harbouring the vector alone (Ms_vec) were generated from the non-pathogenic Mycobacterium smegmatis. These bacterial strains were used to infect peritoneal exudate and bone marrow-derived macrophages. It was found that, despite the comparable intracellular survival between the two recombinant M. smegmatis strains, Ms_ppe37 induced a significantly lower level of tumour necrosis factor alpha and interleukin 6 in the infected macrophages compared with Ms_vec. Western blot analyses revealed that the activation levels of nuclear factor kappa B, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase and MAPK/p38 were lower in macrophages infected with Ms_ppe37 than in macrophages infected with Ms_vec. These results suggest that PPE37 may have a potential role in interfering with the pro-inflammatory cytokine response of infected macrophages. INTRODUCTIONThe existence of the proline-proline-glutamic acid (PPE) multigene family was revealed when the decoding of the Mycobacterium tuberculosis genome was completed (Cole et al., 1998). Members of this gene family were found to share a highly conserved N-terminal sequence of approximately 180 aa that also contained the conserved PPE motif. In contrast, their C-terminal regions were highly heterogeneous in both sequence and length (Cole et al., 1998). The ppe genes are not found outside the genus Mycobacterium and are highly distributed among the pathogenic species of mycobacteria (Gey van Pittius et al., 2006). For these reasons, they are speculated to contribute to the pathogenicity of M. tuberculosis. A possible functional role has been proposed for the PPE proteins, in which they serve as a source of antigenic variation that promotes antigenic diversity in M. tuberculosis (Cole et al., 1998). Indeed, the PPE proteins reported to date are immunogenic, eliciting either humoral or T-cell immune responses (Choudhary et al., 2003; Khan et al., 2008;Romano et al., 2008; Tundup et al., 2008;Wang et al., 2008).One of the PPE proteins, PPE37, may have a role in the virulence of M. tuberculosis. It has been reported that expression of the ppe37 gene is upregulated in M. tuberculosis during infection of murine bone marrowderived macrophages (Schnappinger et al., 2003;Voskuil et al., 2004). Moreover, in M. tuberculosis isolated from the lungs of infected mice, expression of the ppe37 gene is enhanced even more (Schnappinger et al., 2003). Earlier studies have shown that an iron-dependent transcriptional regulator that is critical for proper iron homeosta...

It was shown that virulent Mycobacterium tuberculosis H37Rv induces necrosis of infected RAW264 cells at 24 h post infection while avirulent H37Ra and an attenuated H37Rv mutant that is deficient for RD1 region (H37RvDeltaRD1) cause less necrosis of the infected cells. While H37Rv caused damage of the mitochondrial inner membrane and decreased the level of intracellular ATP, H37RvDeltaRD1 did not exhibit these harmful effects in infected cells. On the other hand, there was no difference in the level of intracellular reactive oxygen species after infection with H37Rv or H37RvDeltaRD1, and the intracellular bacterial numbers of H37RvDeltaRD1 and H37Ra were comparable to that of H37Rv. These results suggested that some virulence factors of H37Rv may contribute to the necrosis of infected cells through induction of mitochondrial dysfunction and depletion of intracellular ATP. RD1 appeared to encode some components possibly playing a central role in the induction of host cell necrosis after M. tuberculosis infection.