- Email: [email protected]
ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection
Medical Hypotheses 78 (2012) 389–392
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection Si Guo a,1, Rui Xue b,1, Yi Li a, Shan Mei Wang a, Lin Ren c, Jing Jing Xu d,⇑ a
Medical Microbiology Laboratory, Henan Provincial People’s Hospital, 7 Wei Wu Road, Zhengzhou 450000, People’s Republic of China Institute of clinical medicine, The First Affiliated Hospital of Zhengzhou University, 1 Jian She Dong Avenue, Zhengzhou 450002, People’s Republic of China c Department of Infection Control, Henan Provincial People’s Hospital, 7 Wei Wu Road, Zhengzhou 450000, People’s Republic of China d Department of Pathology, The First Affiliated Hospital of Zhengzhou University, 1 Jian She Dong Avenue, Zhengzhou 450002, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 23 May 2011 Accepted 27 November 2011
a b s t r a c t Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus CalmetteGuérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor a (TNF-a) from macrophages. Furthermore, TNF-a can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Tuberculosis causes 2–3 million people death worldwide annually, and Mycobacterium tuberculosis is the etiologic agent of human tuberculosis [1,2]. Macrophages play an important role in host defense against bacterial infection by exerting microbicidal activity. However, M. tuberculosis could survive and replicate in human macrophages through modulating host immune response. There is evidence that induction of infected cell death is a vital and alternative strategy for host defense against M. tuberculosis. For instance, it has been reported that macrophages going into apoptosis upon infection with M. tuberculosis results in the suppression of intracellular bacterial replication [3,4]. Furthermore, Schaible et al. reported that the apoptotic vesicles from infected macrophages have an important role in transporting the mycobacterial antigens to immature dendritic cells and activating the adaptive immunity against M. tuberculosis [5]. Above data suggest ⇑ Corresponding author. Tel.: +86 0371 67966151. 1
E-mail address: [email protected] (J.J. Xu). These authors contributed equally to this manuscript.
0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.11.022
that apoptosis of the infected cells constitute an important part of the host defense and affect the fate of intracellular M. tuberculosis. Further investigation showed that virulent species of mycobacteria induced considerably less apoptosis in macrophages than avirulent mycobacterial species [6]. Several reports also indicated that virulent mycobacterial species could actively suppress or down regulate apoptosis of infected macrophages [3,7,8]. Virulent but not avirulent strains of mycobacteria inhibited apoptosis of infected macrophages, thus we proposed that host cell apoptosis inhibition is one of virulent mechanisms of M. tuberculosis. Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrast, it presents in all virulent members of the M. tuberculosis and Mycobacterium bovis [9]. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by the RD1 genes Rv3874 and Rv3875 of M. tuberculosis [10]. Renshaw et al. demonstrated that CFP10 and ESAT6 might be biological active molecules in vivo when they form a 1:1 heterodimeric complex [11]. CFP10 and ESAT6 play an important role in Mtb virulence. It has been shown that incorporation of the RD1 region into BCG to restore
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the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium [12–14]. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence [15]. Several studies investigated the interaction between the two secreted mycobacterial antigens and its host macrophages. It has been shown that CFP10/ESAT6 complex attenuated the innate immune response by inhibiting production of the IL-12, TNF-a from macrophages [16]. Stanley et al. also reported snm4 (Rv3877) and esat6 gene knock out mutants of M. tuberculosis could enhance TNF-a production from macrophages [17]. On the contrary, Trajkovic et al. reported that CFP10 could induce release of TNF-a from J774 macrophages [18]. In addition, the CFP10/ ESAT6 complex was reported to enhance TNF-a production from human monocytes and THP-1 cells in a dose- and time-dependent manner [19]. These results suggest that the CFP10/ESAT6 complex probably has differential effect on macrophage function, which may be involved in virulence mechanism of M. tuberculosis. The hypothesis Previous study found that the secretion of CFP10 and ESAT6 is critical for M. tuberculosis growth in vivo [17]. Furthermore, we demonstrated that the CFP10/ESAT6 complex of M. tuberculosis did not directly affect the viability and proliferation of macrophages [20]. It has been shown that CFP10/ESAT6 complex could inhibit and/or promote production of TNF-a from macrophages [16,19]. TNF-a produced by macrophages plays complicated dual roles in tuberculosis infection. The cytokine is essential for controlling immune cells recruitment to the granulomas [21]. Meanwhile, TNF-a is a major inducer of apoptosis for macrophage harboring intracellular mycobacteria [22]. Apoptosis of infected macrophages is largely beneficial for the host. The capacity of M. tuberculosis to suppress apoptosis mediated by TNF-a is correlated with virulence of M. tuberculosis; it preserves the intracellular environment favorable for bacilli replication while limiting the antimicrobial effects of apoptosis. On the other hand, necrosis mediated by the cytokine is associated with the lysis of the infected cells. The necrotic cells release viable M. tuberculosis which can reinfect new host cells. Considering above results, we hypothesize that the CFP10/ESAT6 complex of M. tuberculosis may act as a virulent factor, which inhibit and/or induce the death of infected macrophages through modulating production of TNF-a. Discussion M. tuberculosis, the causative agent of tuberculosis, is able to survive and replicate inside macrophages. The induction of macrophage apoptosis after the encounter of pathogenic microorganisms may contribute to host innate immune response. It has been reported that macrophage apoptosis reduces mycobacterial viability, which represents a successful host immune response against this intracellular infection [23–25]. On the other hand, several recent studies showed that virulent mycobacteria could inhibit the apoptosis of host macrophages. To data, the mechanisms of the host cell apoptosis inhibition mediated by M. tuberculosis are poorly understood. It has been shown that virulent species of mycobacteria (MtbH37Rv, M. bovis) induce considerably less macrophage apoptosis than avirulent mycobacterial species (BCG, H37Ra). As apoptotic cell death reduced mycobacterial viability, the capacity of Mtb to inhibit macrophage apoptosis was proposed to be a virulence factor. Analysis of comparative genomics has revealed that the region of difference 1(RD1) of M. tuberculosis is absent from all strains of BCG. On the contrast, it presents in all virulent members
of M. tuberculosis and M. bovis [9]. Recent studies further demonstrated that CFP10 and ESAT6 play an important role in Mtb virulence. These results raise the possibility that CFP10 and ESAT6 are involved in apoptosis inhibition in macrophages infected with M. tuberculosis. Previous published data showed that macrophages infected with avirulent mycobacterial species produced more TNF-a than macrophages infected with virulent M. tuberculosis [26]. It has been reported that ESAT6 or CFP10/ESAT6 complex of virulent M. tuberculosis attenuate the innate immune response by inhibiting production of IL-12, TNF-a from macrophages [16]. Stanley et al. investigated the avirulent mutants of M. tuberculosis with lesions in genes encoding components of a secretion system which is responsible for exporting CFP10 and ESAT6.Results showed that Rv3877 and esat6 gene knock out mutants of M. tuberculosis enhanced production of TNF-a in macrophages [17]. The experimental evidences suggest that ESAT6 and the secretion system exporting CFP10 and ESAT6 are major determinants of M. tuberculosis virulence through suppressing the proinflammatory cytokine release from macrophages. In addition, it has been noted that heightened immune responsiveness to ESAT6 correlated with enhanced expression of IL-4 in tuberculosis [27–29]. Recent work further demonstrated that IL-4 could suppress the expression of TNFa and two TNF-a receptors during mycobacterial infection [30]. We know that the production of the proinflammatory cytokine TNF-a is critical for controlling M. tuberculosis infection. In addition, TNF-a can induce apoptosis via TNF-a receptor1 (TNFR1) signaling. Keane et al. reported that TNF-a is a major initiator of apoptotic signaling for macrophages harboring intracellular mycobacteria [22]. Other studies also showed that intracellular infection by M. tuberculosis and other intracellular bacteria can sensitize various cell types to TNF-a mediated death [31–33]. All of these support the hypothesis that CFP10/ESAT6 complex might inhibit the apoptosis of infected cells through decreasing the production of TNF-a. On the other hand, there are conflicting reports that CFP10 or CFP10/EAT6 complex can induce TNF-a release from J774 macrophages and THP-1 cells [18,19]. The role of TNF-a is essential for recruitment of the immune cells necessary for sealing up infection foci inside granulomas [21]. Flynn et al. demonstrated that mice lacking TNF-a or TNF-a signaling quickly succumb to M. tuberculosis infection with respiratory failure [34,35]. However, excess of TNF-a production may also cause pathology, including necrosis which is associated with enhanced TNF-a level [36– 38]. Meanwhile, several studies indicated that TNF-a could promote the intracellular replication of M. tuberculosis in human monocytes and alveolar macrophages [39,40]. It seems that TNF-a in tuberculosis infection is a double-edged sword. Thus we can not rule out the possibility that CFP10/ESAT6 complex may also participate in sustained TNF-a secretion accompanying the persistence of mycobacteria in infected macrophages and lead to necrosis of host cells. Given these observations, the CFP10/ESAT6 complex seems to have dual abilities to inhibit and/or induce host cell death through modulating expression of TNF-a. The process would be similar to the pathology of Chlamydia and Legionella in macrophages, which both go through an anti-apoptotic phase during earlier stages of infection, and then change into a pro-necrotic phase at later stages [41–43]. Inhibition of host cell apoptosis may give M. tuberculosis a protected intracellular environment favorable for pathogen replication at early infection stages, necrosis of host cells at later stages of the infection would benefit bacteria since necrotic cells are able to release viable M. tuberculosis which could reinfect adjacent cells [44–46].Collectively, the dual influences of CFP10/ESAT6 complex on macrophage function might be one of the virulence mechanisms of M. tuberculosis invasion.
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Although there are data indicate that CFP10/ESAT6 complex is closely associated with virulence of M. tuberculosis, less is known about the mechanism of the complex virulence. Our previous study showed that CFP10/ESAT6 complex could not directly subvert the viability and proliferation of macrophages [20]. Similar results on the cell viability were also observed in U937 cells treated by the CFP10/ESAT6 complex [47]. This suggests that the tuberculosis virulence factor do not directly subvert the innate immune cells but utilize other mechanisms. The hypothesis put forward to explain mechanism of the CFP10/ESAT6 complex involved in Mycobacterium virulence. Our hypothesis does not exclude other possible mechanisms of virulence mediated by the CFP10/ESAT6 complex. It is also possible that interaction between the CFP10/ESAT6 complex with receptors of macrophages triggers cell death signaling pathways of macrophages. Since we and other groups demonstrated the soluble and secreted complex could bind to the surface of macrophages. The hypothesis can be tested by determining whether the complex is truly involved in host cell death through studies on cfp-10 and esat-6 gene knock out mutant of M. tuberculosis in vivo and vitro. This study will offer an evidence of inhibiting and/or inducing macrophage cell death mediated by the CFP10/ESAT6 complex. The confirmation of the hypothesis can lead to a greater understanding of pathogenesis of tuberculosis. Only thorough understanding of virulence mechanisms of M. tuberculosis, it is possible for development of new approaches to treatment tuberculosis. Conflicts of interest statement None declared. Acknowledgement This study was supported by grants from the Youth Foundation of The First Affiliated Hospital of Zhengzhou University. References [1] Dye C, Scheel S, Dolin P, Pathania V, Raviglion MC. Consensus statement. Global burden of tuberculosis estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. JAMA 1999;282:677–86. [2] Raviglione MC, Dye C, Schmidt S, Kochi A. Assessment of worldwide tuberculosis control. WHO global surveillance and monitoring project. Lancet 1997;350:624–9. [3] Riendeau CJ, Kornfeld H. THP-1 cell apoptosis in response to mycobacterium infection. Infect Immun 2003;71:254–9. [4] Sly LM, Hingley-Wilson SM, Reiner NE, McMaster WR. Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1. J Immunol 2003;170:430–7. [5] Schaible UE, Winau F, Sieling PA, et al. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 2003;9:1039–46. [6] Keane J, Remold HG, Kornfled H. Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophage. J Immunol 2000;164:2016–20. [7] Dhiman R, Raje M, Majumdar S. Differential expression of NF-kappa B in mycobacteria infected THP-1 affects apoptosis. Biochim Biophys Acta 2007;1770:649–58. [8] Zhang J, Jiang R, Takayama H, Tanaka Y. Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages. Microbiol Immunol 2005;49:845–52. [9] Behr MA, Wilson MA, Gill WP, et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 1999;284:1520–3. [10] Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB. Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med 1999;189:1973–80. [11] Renshaw PS, Panagiotidou P, Whelan A, et al. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6-CFP-10 complex: implications for pathogenesis and virulence. J Biol Chem 2002;277:21598–603.
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[17] Stanely S, Raghavan S, Hwang WW, Cox JS. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci 2003;100:13001–6. [18] Trajkovic G, Singh G, Singh B, et al. Effect of Mycobacterium tuberculosis 10Kilodalton antigen on macrophage release of tumor necrosis factor alpha and nitric oxide. Infect Immun 2002;70:6558–66. [19] Feng Y, Yang X, Liu Z, et al. Continuous treatment with recombinant Mycobacterium tuberculosis CFP-10-ESAT-6 protein activated human monocyte while deactivated LPS-stimulated macrophage. Biochem Biophys Res Commun 2008;365:534–40. [20] Si G, Lang B, Zi Fang Q, Xin Xin S. The CFP10/ESAT6 complex of Mycobacterium tuberculosis potentiates the activation of murine macrophages involvement of IFN-c signaling. Med Microbiol Immunol 2010;991:129–37. [21] Kindler V, Sappino AP, Grau GE, Piguet PF, Vassalli P. 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Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection Si Guo a,1, Rui Xue b,1, Yi Li a, Shan Mei Wang a, Lin Ren c, Jing Jing Xu d,⇑ a
Medical Microbiology Laboratory, Henan Provincial People’s Hospital, 7 Wei Wu Road, Zhengzhou 450000, People’s Republic of China Institute of clinical medicine, The First Affiliated Hospital of Zhengzhou University, 1 Jian She Dong Avenue, Zhengzhou 450002, People’s Republic of China c Department of Infection Control, Henan Provincial People’s Hospital, 7 Wei Wu Road, Zhengzhou 450000, People’s Republic of China d Department of Pathology, The First Affiliated Hospital of Zhengzhou University, 1 Jian She Dong Avenue, Zhengzhou 450002, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 23 May 2011 Accepted 27 November 2011
a b s t r a c t Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus CalmetteGuérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor a (TNF-a) from macrophages. Furthermore, TNF-a can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Tuberculosis causes 2–3 million people death worldwide annually, and Mycobacterium tuberculosis is the etiologic agent of human tuberculosis [1,2]. Macrophages play an important role in host defense against bacterial infection by exerting microbicidal activity. However, M. tuberculosis could survive and replicate in human macrophages through modulating host immune response. There is evidence that induction of infected cell death is a vital and alternative strategy for host defense against M. tuberculosis. For instance, it has been reported that macrophages going into apoptosis upon infection with M. tuberculosis results in the suppression of intracellular bacterial replication [3,4]. Furthermore, Schaible et al. reported that the apoptotic vesicles from infected macrophages have an important role in transporting the mycobacterial antigens to immature dendritic cells and activating the adaptive immunity against M. tuberculosis [5]. Above data suggest ⇑ Corresponding author. Tel.: +86 0371 67966151. 1
E-mail address: [email protected] (J.J. Xu). These authors contributed equally to this manuscript.
0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.11.022
that apoptosis of the infected cells constitute an important part of the host defense and affect the fate of intracellular M. tuberculosis. Further investigation showed that virulent species of mycobacteria induced considerably less apoptosis in macrophages than avirulent mycobacterial species [6]. Several reports also indicated that virulent mycobacterial species could actively suppress or down regulate apoptosis of infected macrophages [3,7,8]. Virulent but not avirulent strains of mycobacteria inhibited apoptosis of infected macrophages, thus we proposed that host cell apoptosis inhibition is one of virulent mechanisms of M. tuberculosis. Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrast, it presents in all virulent members of the M. tuberculosis and Mycobacterium bovis [9]. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by the RD1 genes Rv3874 and Rv3875 of M. tuberculosis [10]. Renshaw et al. demonstrated that CFP10 and ESAT6 might be biological active molecules in vivo when they form a 1:1 heterodimeric complex [11]. CFP10 and ESAT6 play an important role in Mtb virulence. It has been shown that incorporation of the RD1 region into BCG to restore
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the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium [12–14]. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence [15]. Several studies investigated the interaction between the two secreted mycobacterial antigens and its host macrophages. It has been shown that CFP10/ESAT6 complex attenuated the innate immune response by inhibiting production of the IL-12, TNF-a from macrophages [16]. Stanley et al. also reported snm4 (Rv3877) and esat6 gene knock out mutants of M. tuberculosis could enhance TNF-a production from macrophages [17]. On the contrary, Trajkovic et al. reported that CFP10 could induce release of TNF-a from J774 macrophages [18]. In addition, the CFP10/ ESAT6 complex was reported to enhance TNF-a production from human monocytes and THP-1 cells in a dose- and time-dependent manner [19]. These results suggest that the CFP10/ESAT6 complex probably has differential effect on macrophage function, which may be involved in virulence mechanism of M. tuberculosis. The hypothesis Previous study found that the secretion of CFP10 and ESAT6 is critical for M. tuberculosis growth in vivo [17]. Furthermore, we demonstrated that the CFP10/ESAT6 complex of M. tuberculosis did not directly affect the viability and proliferation of macrophages [20]. It has been shown that CFP10/ESAT6 complex could inhibit and/or promote production of TNF-a from macrophages [16,19]. TNF-a produced by macrophages plays complicated dual roles in tuberculosis infection. The cytokine is essential for controlling immune cells recruitment to the granulomas [21]. Meanwhile, TNF-a is a major inducer of apoptosis for macrophage harboring intracellular mycobacteria [22]. Apoptosis of infected macrophages is largely beneficial for the host. The capacity of M. tuberculosis to suppress apoptosis mediated by TNF-a is correlated with virulence of M. tuberculosis; it preserves the intracellular environment favorable for bacilli replication while limiting the antimicrobial effects of apoptosis. On the other hand, necrosis mediated by the cytokine is associated with the lysis of the infected cells. The necrotic cells release viable M. tuberculosis which can reinfect new host cells. Considering above results, we hypothesize that the CFP10/ESAT6 complex of M. tuberculosis may act as a virulent factor, which inhibit and/or induce the death of infected macrophages through modulating production of TNF-a. Discussion M. tuberculosis, the causative agent of tuberculosis, is able to survive and replicate inside macrophages. The induction of macrophage apoptosis after the encounter of pathogenic microorganisms may contribute to host innate immune response. It has been reported that macrophage apoptosis reduces mycobacterial viability, which represents a successful host immune response against this intracellular infection [23–25]. On the other hand, several recent studies showed that virulent mycobacteria could inhibit the apoptosis of host macrophages. To data, the mechanisms of the host cell apoptosis inhibition mediated by M. tuberculosis are poorly understood. It has been shown that virulent species of mycobacteria (MtbH37Rv, M. bovis) induce considerably less macrophage apoptosis than avirulent mycobacterial species (BCG, H37Ra). As apoptotic cell death reduced mycobacterial viability, the capacity of Mtb to inhibit macrophage apoptosis was proposed to be a virulence factor. Analysis of comparative genomics has revealed that the region of difference 1(RD1) of M. tuberculosis is absent from all strains of BCG. On the contrast, it presents in all virulent members
of M. tuberculosis and M. bovis [9]. Recent studies further demonstrated that CFP10 and ESAT6 play an important role in Mtb virulence. These results raise the possibility that CFP10 and ESAT6 are involved in apoptosis inhibition in macrophages infected with M. tuberculosis. Previous published data showed that macrophages infected with avirulent mycobacterial species produced more TNF-a than macrophages infected with virulent M. tuberculosis [26]. It has been reported that ESAT6 or CFP10/ESAT6 complex of virulent M. tuberculosis attenuate the innate immune response by inhibiting production of IL-12, TNF-a from macrophages [16]. Stanley et al. investigated the avirulent mutants of M. tuberculosis with lesions in genes encoding components of a secretion system which is responsible for exporting CFP10 and ESAT6.Results showed that Rv3877 and esat6 gene knock out mutants of M. tuberculosis enhanced production of TNF-a in macrophages [17]. The experimental evidences suggest that ESAT6 and the secretion system exporting CFP10 and ESAT6 are major determinants of M. tuberculosis virulence through suppressing the proinflammatory cytokine release from macrophages. In addition, it has been noted that heightened immune responsiveness to ESAT6 correlated with enhanced expression of IL-4 in tuberculosis [27–29]. Recent work further demonstrated that IL-4 could suppress the expression of TNFa and two TNF-a receptors during mycobacterial infection [30]. We know that the production of the proinflammatory cytokine TNF-a is critical for controlling M. tuberculosis infection. In addition, TNF-a can induce apoptosis via TNF-a receptor1 (TNFR1) signaling. Keane et al. reported that TNF-a is a major initiator of apoptotic signaling for macrophages harboring intracellular mycobacteria [22]. Other studies also showed that intracellular infection by M. tuberculosis and other intracellular bacteria can sensitize various cell types to TNF-a mediated death [31–33]. All of these support the hypothesis that CFP10/ESAT6 complex might inhibit the apoptosis of infected cells through decreasing the production of TNF-a. On the other hand, there are conflicting reports that CFP10 or CFP10/EAT6 complex can induce TNF-a release from J774 macrophages and THP-1 cells [18,19]. The role of TNF-a is essential for recruitment of the immune cells necessary for sealing up infection foci inside granulomas [21]. Flynn et al. demonstrated that mice lacking TNF-a or TNF-a signaling quickly succumb to M. tuberculosis infection with respiratory failure [34,35]. However, excess of TNF-a production may also cause pathology, including necrosis which is associated with enhanced TNF-a level [36– 38]. Meanwhile, several studies indicated that TNF-a could promote the intracellular replication of M. tuberculosis in human monocytes and alveolar macrophages [39,40]. It seems that TNF-a in tuberculosis infection is a double-edged sword. Thus we can not rule out the possibility that CFP10/ESAT6 complex may also participate in sustained TNF-a secretion accompanying the persistence of mycobacteria in infected macrophages and lead to necrosis of host cells. Given these observations, the CFP10/ESAT6 complex seems to have dual abilities to inhibit and/or induce host cell death through modulating expression of TNF-a. The process would be similar to the pathology of Chlamydia and Legionella in macrophages, which both go through an anti-apoptotic phase during earlier stages of infection, and then change into a pro-necrotic phase at later stages [41–43]. Inhibition of host cell apoptosis may give M. tuberculosis a protected intracellular environment favorable for pathogen replication at early infection stages, necrosis of host cells at later stages of the infection would benefit bacteria since necrotic cells are able to release viable M. tuberculosis which could reinfect adjacent cells [44–46].Collectively, the dual influences of CFP10/ESAT6 complex on macrophage function might be one of the virulence mechanisms of M. tuberculosis invasion.
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Although there are data indicate that CFP10/ESAT6 complex is closely associated with virulence of M. tuberculosis, less is known about the mechanism of the complex virulence. Our previous study showed that CFP10/ESAT6 complex could not directly subvert the viability and proliferation of macrophages [20]. Similar results on the cell viability were also observed in U937 cells treated by the CFP10/ESAT6 complex [47]. This suggests that the tuberculosis virulence factor do not directly subvert the innate immune cells but utilize other mechanisms. The hypothesis put forward to explain mechanism of the CFP10/ESAT6 complex involved in Mycobacterium virulence. Our hypothesis does not exclude other possible mechanisms of virulence mediated by the CFP10/ESAT6 complex. It is also possible that interaction between the CFP10/ESAT6 complex with receptors of macrophages triggers cell death signaling pathways of macrophages. Since we and other groups demonstrated the soluble and secreted complex could bind to the surface of macrophages. The hypothesis can be tested by determining whether the complex is truly involved in host cell death through studies on cfp-10 and esat-6 gene knock out mutant of M. tuberculosis in vivo and vitro. This study will offer an evidence of inhibiting and/or inducing macrophage cell death mediated by the CFP10/ESAT6 complex. The confirmation of the hypothesis can lead to a greater understanding of pathogenesis of tuberculosis. Only thorough understanding of virulence mechanisms of M. tuberculosis, it is possible for development of new approaches to treatment tuberculosis. Conflicts of interest statement None declared. Acknowledgement This study was supported by grants from the Youth Foundation of The First Affiliated Hospital of Zhengzhou University. References [1] Dye C, Scheel S, Dolin P, Pathania V, Raviglion MC. Consensus statement. Global burden of tuberculosis estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. JAMA 1999;282:677–86. [2] Raviglione MC, Dye C, Schmidt S, Kochi A. Assessment of worldwide tuberculosis control. WHO global surveillance and monitoring project. Lancet 1997;350:624–9. [3] Riendeau CJ, Kornfeld H. THP-1 cell apoptosis in response to mycobacterium infection. Infect Immun 2003;71:254–9. [4] Sly LM, Hingley-Wilson SM, Reiner NE, McMaster WR. Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1. J Immunol 2003;170:430–7. [5] Schaible UE, Winau F, Sieling PA, et al. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 2003;9:1039–46. [6] Keane J, Remold HG, Kornfled H. Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophage. J Immunol 2000;164:2016–20. [7] Dhiman R, Raje M, Majumdar S. Differential expression of NF-kappa B in mycobacteria infected THP-1 affects apoptosis. Biochim Biophys Acta 2007;1770:649–58. [8] Zhang J, Jiang R, Takayama H, Tanaka Y. Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages. Microbiol Immunol 2005;49:845–52. [9] Behr MA, Wilson MA, Gill WP, et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 1999;284:1520–3. [10] Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB. Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med 1999;189:1973–80. [11] Renshaw PS, Panagiotidou P, Whelan A, et al. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6-CFP-10 complex: implications for pathogenesis and virulence. J Biol Chem 2002;277:21598–603.
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