The Mycobacterium tuberculosis ESAT-6 Homologue in Listeria monocytogenes Is Dispensable for Growth In Vitro and In Vivo
http://www.100md.com
感染与免疫杂志 2005年第9期
Departments of Pediatrics Immunology, University of Washington School of Medicine, Seattle, Washington 98195
ABSTRACT
ESAT-6 is a virulence determinant in Mycobacterium tuberculosis and a member of a conserved group of proteins in a variety of other bacteria. A targeted deletion of the homologous gene in Listeria was generated, and in contrast to that observed for mycobacteria, this locus was not required for Listeria virulence.
TEXT
Infection with Mycobacterium tuberculosis is a major global public health problem. Over one-third of the world's population is infected, and an estimated 2 million deaths occur each year as a result. Comparative genome analysis between the nonpathogenic mycobacterium bacille Calmette-Guerin and the pathogenic mycobacterium M. tuberculosis or Mycobacterium bovis has revealed a 9.5-kb region (RD1) to be important for virulence (1, 2, 9). Spontaneous deletion of this region is thought to be the primary mutation that occurred in M. bovis during serial passage by Calmette and Guerin between 1908 and 1921, resulting in the current widely used vaccine strain BCG. This hypothesis is supported by the severe in vivo attenuation of M. tuberculosis after targeted deletion of this region and restoration of virulence by complementation of this region in BCG (7, 8, 13).
In M. tuberculosis, 11 open reading frames (ORFs) are present in the 9.5-kb RD1 region. Three of these ORFs encode critical components of a secretion system that exports two additional protein products encoded within RD1, ESAT-6 and CFP-10 (14). Both ESAT-6 and CFP-10 contain immunodominant epitopes in the T-cell response to M. tuberculosis infection (12). Although the mechanism of action of ESAT-6 is unknown, this specific locus is essential for mycobacterial virulence because targeted deletion of this gene in either M. tuberculosis or M. bovis results in profound attenuation following in vivo infection (14, 15).
Homologues of the M. tuberculosis ESAT-6 protein have been identified in a variety of other bacterial species. While initial studies identified homologues in high-G+C bacterial species such as actinobacteria and other mycobacteria (6), further analyses have identified more distant homologues in various species of gram-positive bacteria in the low-G+C group, including Bacillus spp., Clostridium spp., Staphylococcus spp., and Listeria spp. (10). Although the level of sequence similarity of these proteins compared with ESAT-6 is relatively low (30% similarity and 15% identity), conservation of the internal tryptophan-X-glycine (W-X-G) and 100-residue length supports the inclusion of these proteins in an ESAT-6 superfamily of "WXG100" proteins. Recently, the homologue of M. tuberculosis ESAT6 in Staphylococcus aureus has been identified and found to play an essential role in S. aureus virulence following intravenous infection (3).
Listeria monocytogenes is a gram-positive bacterium that causes human clinical disease ranging from self-resolving gastroenteritis to more serious illnesses, including abortion, sepsis, and central nervous system infection. Neonates and other immunocompromised hosts are especially susceptible to more serious infection. The pathogenesis and virulence determinants of L. monocytogenes have been well characterized with in vitro and in vivo infection models (4). During infection, L. monocytogenes coordinates the expression of an array of bacterial gene products to gain access and reside within the intracytoplasmic compartment of infected cells, thereby evading humoral immunity, and as a result protective immunity to this infection is mediated predominantly by cellular mediators (11). L. monocytogenes infection is currently widely used as a model to study and identify host mediators of innate and adaptive immunity to intracellular bacterial pathogens.
The L. monocytogenes ESAT-6 homologue is a 99-residue protein and is encoded by the lm00056 locus (Lmesat6) identified by genome sequencing (National Center for Biotechnology Information accession no. AL591973). At the protein level, it is 14% identical and 30% similar to M. tuberculosis ESAT-6 and contains the conserved W-X-G motif (Fig. 1A). During infection, transcription of this locus can be detected in the spleens of infected mice by reverse transcription-PCR (Fig. 1B). The importance of this protein in the virulence of other bacterial and mycobacterial pathogens and the conserved nature of homologous proteins in diverse bacterial species suggested they may play a conserved role in bacterial replication and/or pathogenesis. Thus, we sought to examine the role of this locus in L. monocytogenes infections by comparing the in vitro and in vivo virulence properties of an Lmesat6 deletion mutant with wild-type (WT) L. monocytogenes. A targeted internal 170-bp deletion within the Lmesat6 open reading frame was generated by homologous recombination after cloning upstream and downstream fragments into the temperature-sensitive construct pKSV7 (Fig. 1C and D) and electroporation into WT L. monocytogenes strain 10403s using previously described methods (5).
The growth and virulence properties of this mutant, L. monocytogenes esat6, were compared with the parental WT L. monocytogenes strain. The growth rates of this mutant in cell-free medium under both aerobic (300 rpm on an orbital shaker) and microaerophilic (soft agar) conditions were identical to those of the parental strain. Similarly, the size and frequency of bacterial plaques formed after in vitro infection of HeLa cell monolayers were identical between the mutant and parental strains (data not shown).
We further evaluated the importance of Lmesat6 during in vivo infection. Following intravenous infection with either WT L. monocytogenes or L. monocytogenes esat6, bacterial replication uniformly occurred in the spleens and liver at day 3 compared with the initial inocula, and bacterial clearance uniformly occurred by day 7 (Fig. 2). However, at each of these time points, no significant differences in bacterial burden were detected in either organ between mice infected with L. monocytogenes esat6 and WT L. monocytogenes. In the time course of primary L. monocytogenes infection, innate immune cells such as macrophages, neutrophils, and natural killer cells are responsible for control of bacterial replication in the first 5 days following infection, while immunity thereafter is mediated by antigen-specific CD8 and CD4 T cells. Thus, the normal bacterial clearance from day 3 to day 7 following infection by L. monocytogenes esat6-infected mice suggests that the L. monocytogenes homologue of ESAT6 is not required for generation of L. monocytogenes antigen-specific CD8 and CD4 T cells. This was tested by examining the percentage and total numbers of L. monocytogenes-specific CD8 and CD4 T cells after infection with either WT L. monocytogenes or L. monocytogenes esat6. At the peak of the L. monocytogenes-specific T-cell response (day 7), splenocytes from infected mice were stimulated with either the major histocompatibility complex (MHC) class I antigenic peptide LLO 91-99 (CD8 T cells) or the class II peptide LLO 189-201 (CD4 T cells) and specific cells were quantified by cell surface and intracellular cytokine staining as previously described (16) (Fig. 3). While infection with both strains elicited a robust CD8 and CD4 T-cell response, there was no difference in either the percentage or the total number of L. monocytogenes-specific CD8 or CD4 T cells after infection with L. monocytogenes esat6 or WT L. monocytogenes.
These data demonstrate that the L. monocytogenes homologue to M. tuberculosis ESAT6 does not play a detectable role in L. monocytogenes virulence or in the triggering of L. monocytogenes-specific adaptive T-cell-mediated immune responses. The lack of an obvious role for L. monocytogenes ESAT-6 could be due to the intracytoplasmic residence of L. monocytogenes compared with the intravacuolar residence of Mycobacterium spp. and Staphylococcus spp. or other intrinsic differences between the virulence properties of these bacterial pathogens. The data presented in this report extend our knowledge regarding the function of the WXG100 protein family in bacterial virulence by showing that it is not required in all contexts. Thus, further evaluation of this protein family's role in the virulence of other bacterial pathogens is warranted.
ACKNOWLEDGMENTS
We acknowledge Ester Li, Carleen Collins, Nancy Freitag, Adeline Hajjar, Tobias Kollmann, David Sherman, and Kevin Urdahl for helpful discussion and critical review of the manuscript.
This work was supported by NIH grant HD18184 (to C.B.W.). S.S.W. is an NICHD Fellow of the Pediatric Scientist Development Program (NICHD grant award K12-HD00850).
REFERENCES
1. Behr, M. A., M. A. Wilson, W. P. Gill, H. Salamon, G. K. Schoolnik, S. Rane, and P. M Small. 1999. Comparative genomics of BCG vaccines by whole genome DNA microarray. Science 284:1520-1523.
2. Brosch, R., S. V. Gordon, C. Buchrieser, A. S. Pym, T. Garnier, and S. T. Cole. 2000. Comparative genomics uncovers large tandem chromosomal duplications in the Mycobacterium bovis BCG Pasteur. Yeast 17:111-123.
3. Burts, M. L., W. A. Williams, K. DeBord, and D. M. Missiakas. 2005. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc. Natl. Acad. Sci. USA 102:1169-1174.
4. Cossart, P., and P. J. Sansonetti. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304:242-248.
5. Freitag, N. E. 2000. Genetic tools for use with Listeria monocytogenes, p. 488-498. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood. (ed.), Gram-positive pathogens. ASM Press, Washington, D.C.
6. Gey van Pittius, N. C., J. Gamieldien, W. Hide, G. D. Brown, R. J. Siezen, and A. D. Beyers. 2001. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C gram positive bacteria. Genome Biol. 2:0044.1-0044.18.
7. Hsu, T., S. M. Hingley-Wilson, B. Chen, M. Chen, A. Z. Dai, P. M. Morin, C. B. Marks, J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R. G. Russell, S. C. Derrick, F. M. Collins, S. L. Morris, C. H. King, and W. R. Jacobs. 2003. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA 100:12420-12425.
8. Lewis, K. N., R. Liao, K. M Guinn, M. J. Hickey, S. Smith, M. A. Behr, and D. R. Sherman. 2003. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187:117-123.
9. Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover. 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274-1282.
10. Pallen, M. J. 2002. The ESAT-6/WXG100 superfamily—and a new gram-positive secretion system Trends Microbiol. 10:209-212.
11. Pamer, E. G. 2004. Immune response to Listeria monocytogenes. Nat Rev. Immunol. 4:812-823.
12. Pym, A. S., P. Brodin, L. Majlessi, R. Brosch, C. Demangel, A. Willians, K. E. Griffiths, G. Marchal, C. Leclerc, and S. T. Cole. 2003. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat. Immunol 9:533-539.
13. Pym, A. S., P. Brodin, R. Brosch, M. Huerre, and S. T. Cole. 2002. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46:709-717.
14. Stanley, S. A., S. Raghaven, W. W. Hwang, and J. S. Cox. 2003. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. USA 100:13001-13006.
15. Wards, B. J., G. W. de Lisle, and D. M. Collins. 2000. An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine. Tuber. Lung Dis. 80:185-189.
16. Way, S. S., L. J. Thompson, J. E. Lopes, A. M. Hajjar, T. R. Kollmann, N. E. Freitag, and C. B. Wilson. 2004. Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity. Cell. Microbiol. 6:235-242.(Sing Sing Way and Christo)
ABSTRACT
ESAT-6 is a virulence determinant in Mycobacterium tuberculosis and a member of a conserved group of proteins in a variety of other bacteria. A targeted deletion of the homologous gene in Listeria was generated, and in contrast to that observed for mycobacteria, this locus was not required for Listeria virulence.
TEXT
Infection with Mycobacterium tuberculosis is a major global public health problem. Over one-third of the world's population is infected, and an estimated 2 million deaths occur each year as a result. Comparative genome analysis between the nonpathogenic mycobacterium bacille Calmette-Guerin and the pathogenic mycobacterium M. tuberculosis or Mycobacterium bovis has revealed a 9.5-kb region (RD1) to be important for virulence (1, 2, 9). Spontaneous deletion of this region is thought to be the primary mutation that occurred in M. bovis during serial passage by Calmette and Guerin between 1908 and 1921, resulting in the current widely used vaccine strain BCG. This hypothesis is supported by the severe in vivo attenuation of M. tuberculosis after targeted deletion of this region and restoration of virulence by complementation of this region in BCG (7, 8, 13).
In M. tuberculosis, 11 open reading frames (ORFs) are present in the 9.5-kb RD1 region. Three of these ORFs encode critical components of a secretion system that exports two additional protein products encoded within RD1, ESAT-6 and CFP-10 (14). Both ESAT-6 and CFP-10 contain immunodominant epitopes in the T-cell response to M. tuberculosis infection (12). Although the mechanism of action of ESAT-6 is unknown, this specific locus is essential for mycobacterial virulence because targeted deletion of this gene in either M. tuberculosis or M. bovis results in profound attenuation following in vivo infection (14, 15).
Homologues of the M. tuberculosis ESAT-6 protein have been identified in a variety of other bacterial species. While initial studies identified homologues in high-G+C bacterial species such as actinobacteria and other mycobacteria (6), further analyses have identified more distant homologues in various species of gram-positive bacteria in the low-G+C group, including Bacillus spp., Clostridium spp., Staphylococcus spp., and Listeria spp. (10). Although the level of sequence similarity of these proteins compared with ESAT-6 is relatively low (30% similarity and 15% identity), conservation of the internal tryptophan-X-glycine (W-X-G) and 100-residue length supports the inclusion of these proteins in an ESAT-6 superfamily of "WXG100" proteins. Recently, the homologue of M. tuberculosis ESAT6 in Staphylococcus aureus has been identified and found to play an essential role in S. aureus virulence following intravenous infection (3).
Listeria monocytogenes is a gram-positive bacterium that causes human clinical disease ranging from self-resolving gastroenteritis to more serious illnesses, including abortion, sepsis, and central nervous system infection. Neonates and other immunocompromised hosts are especially susceptible to more serious infection. The pathogenesis and virulence determinants of L. monocytogenes have been well characterized with in vitro and in vivo infection models (4). During infection, L. monocytogenes coordinates the expression of an array of bacterial gene products to gain access and reside within the intracytoplasmic compartment of infected cells, thereby evading humoral immunity, and as a result protective immunity to this infection is mediated predominantly by cellular mediators (11). L. monocytogenes infection is currently widely used as a model to study and identify host mediators of innate and adaptive immunity to intracellular bacterial pathogens.
The L. monocytogenes ESAT-6 homologue is a 99-residue protein and is encoded by the lm00056 locus (Lmesat6) identified by genome sequencing (National Center for Biotechnology Information accession no. AL591973). At the protein level, it is 14% identical and 30% similar to M. tuberculosis ESAT-6 and contains the conserved W-X-G motif (Fig. 1A). During infection, transcription of this locus can be detected in the spleens of infected mice by reverse transcription-PCR (Fig. 1B). The importance of this protein in the virulence of other bacterial and mycobacterial pathogens and the conserved nature of homologous proteins in diverse bacterial species suggested they may play a conserved role in bacterial replication and/or pathogenesis. Thus, we sought to examine the role of this locus in L. monocytogenes infections by comparing the in vitro and in vivo virulence properties of an Lmesat6 deletion mutant with wild-type (WT) L. monocytogenes. A targeted internal 170-bp deletion within the Lmesat6 open reading frame was generated by homologous recombination after cloning upstream and downstream fragments into the temperature-sensitive construct pKSV7 (Fig. 1C and D) and electroporation into WT L. monocytogenes strain 10403s using previously described methods (5).
The growth and virulence properties of this mutant, L. monocytogenes esat6, were compared with the parental WT L. monocytogenes strain. The growth rates of this mutant in cell-free medium under both aerobic (300 rpm on an orbital shaker) and microaerophilic (soft agar) conditions were identical to those of the parental strain. Similarly, the size and frequency of bacterial plaques formed after in vitro infection of HeLa cell monolayers were identical between the mutant and parental strains (data not shown).
We further evaluated the importance of Lmesat6 during in vivo infection. Following intravenous infection with either WT L. monocytogenes or L. monocytogenes esat6, bacterial replication uniformly occurred in the spleens and liver at day 3 compared with the initial inocula, and bacterial clearance uniformly occurred by day 7 (Fig. 2). However, at each of these time points, no significant differences in bacterial burden were detected in either organ between mice infected with L. monocytogenes esat6 and WT L. monocytogenes. In the time course of primary L. monocytogenes infection, innate immune cells such as macrophages, neutrophils, and natural killer cells are responsible for control of bacterial replication in the first 5 days following infection, while immunity thereafter is mediated by antigen-specific CD8 and CD4 T cells. Thus, the normal bacterial clearance from day 3 to day 7 following infection by L. monocytogenes esat6-infected mice suggests that the L. monocytogenes homologue of ESAT6 is not required for generation of L. monocytogenes antigen-specific CD8 and CD4 T cells. This was tested by examining the percentage and total numbers of L. monocytogenes-specific CD8 and CD4 T cells after infection with either WT L. monocytogenes or L. monocytogenes esat6. At the peak of the L. monocytogenes-specific T-cell response (day 7), splenocytes from infected mice were stimulated with either the major histocompatibility complex (MHC) class I antigenic peptide LLO 91-99 (CD8 T cells) or the class II peptide LLO 189-201 (CD4 T cells) and specific cells were quantified by cell surface and intracellular cytokine staining as previously described (16) (Fig. 3). While infection with both strains elicited a robust CD8 and CD4 T-cell response, there was no difference in either the percentage or the total number of L. monocytogenes-specific CD8 or CD4 T cells after infection with L. monocytogenes esat6 or WT L. monocytogenes.
These data demonstrate that the L. monocytogenes homologue to M. tuberculosis ESAT6 does not play a detectable role in L. monocytogenes virulence or in the triggering of L. monocytogenes-specific adaptive T-cell-mediated immune responses. The lack of an obvious role for L. monocytogenes ESAT-6 could be due to the intracytoplasmic residence of L. monocytogenes compared with the intravacuolar residence of Mycobacterium spp. and Staphylococcus spp. or other intrinsic differences between the virulence properties of these bacterial pathogens. The data presented in this report extend our knowledge regarding the function of the WXG100 protein family in bacterial virulence by showing that it is not required in all contexts. Thus, further evaluation of this protein family's role in the virulence of other bacterial pathogens is warranted.
ACKNOWLEDGMENTS
We acknowledge Ester Li, Carleen Collins, Nancy Freitag, Adeline Hajjar, Tobias Kollmann, David Sherman, and Kevin Urdahl for helpful discussion and critical review of the manuscript.
This work was supported by NIH grant HD18184 (to C.B.W.). S.S.W. is an NICHD Fellow of the Pediatric Scientist Development Program (NICHD grant award K12-HD00850).
REFERENCES
1. Behr, M. A., M. A. Wilson, W. P. Gill, H. Salamon, G. K. Schoolnik, S. Rane, and P. M Small. 1999. Comparative genomics of BCG vaccines by whole genome DNA microarray. Science 284:1520-1523.
2. Brosch, R., S. V. Gordon, C. Buchrieser, A. S. Pym, T. Garnier, and S. T. Cole. 2000. Comparative genomics uncovers large tandem chromosomal duplications in the Mycobacterium bovis BCG Pasteur. Yeast 17:111-123.
3. Burts, M. L., W. A. Williams, K. DeBord, and D. M. Missiakas. 2005. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc. Natl. Acad. Sci. USA 102:1169-1174.
4. Cossart, P., and P. J. Sansonetti. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304:242-248.
5. Freitag, N. E. 2000. Genetic tools for use with Listeria monocytogenes, p. 488-498. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood. (ed.), Gram-positive pathogens. ASM Press, Washington, D.C.
6. Gey van Pittius, N. C., J. Gamieldien, W. Hide, G. D. Brown, R. J. Siezen, and A. D. Beyers. 2001. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C gram positive bacteria. Genome Biol. 2:0044.1-0044.18.
7. Hsu, T., S. M. Hingley-Wilson, B. Chen, M. Chen, A. Z. Dai, P. M. Morin, C. B. Marks, J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R. G. Russell, S. C. Derrick, F. M. Collins, S. L. Morris, C. H. King, and W. R. Jacobs. 2003. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA 100:12420-12425.
8. Lewis, K. N., R. Liao, K. M Guinn, M. J. Hickey, S. Smith, M. A. Behr, and D. R. Sherman. 2003. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187:117-123.
9. Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover. 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274-1282.
10. Pallen, M. J. 2002. The ESAT-6/WXG100 superfamily—and a new gram-positive secretion system Trends Microbiol. 10:209-212.
11. Pamer, E. G. 2004. Immune response to Listeria monocytogenes. Nat Rev. Immunol. 4:812-823.
12. Pym, A. S., P. Brodin, L. Majlessi, R. Brosch, C. Demangel, A. Willians, K. E. Griffiths, G. Marchal, C. Leclerc, and S. T. Cole. 2003. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat. Immunol 9:533-539.
13. Pym, A. S., P. Brodin, R. Brosch, M. Huerre, and S. T. Cole. 2002. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46:709-717.
14. Stanley, S. A., S. Raghaven, W. W. Hwang, and J. S. Cox. 2003. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. USA 100:13001-13006.
15. Wards, B. J., G. W. de Lisle, and D. M. Collins. 2000. An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine. Tuber. Lung Dis. 80:185-189.
16. Way, S. S., L. J. Thompson, J. E. Lopes, A. M. Hajjar, T. R. Kollmann, N. E. Freitag, and C. B. Wilson. 2004. Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity. Cell. Microbiol. 6:235-242.(Sing Sing Way and Christo)