Intracellular Survival of Campylobacter jejuni in Human Monocytic Cells and Induction of Apoptotic Death by Cytholethal Distending Toxin
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感染与免疫杂志 2005年第8期
Enteric Diseases Department, Naval Medical Research Center, Silver Spring, Maryland 20910
ABSTRACT
Campylobacter jejuni 81-176 is capable of extensive replication within human monocytic cell vacuoles and induces apoptotic death via cytolethal distending toxin.
TEXT
Our understanding of the interaction of Campylobacter jejuni with immune effector cells is limited. C. jejuni can survive phagocytosis by human mononuclear cells and remain viable for extended periods (9), induce secretion of chemokines (8), and mediate apoptosis (20). C. jejuni elaborates a well-characterized toxin, cytolethal distending toxin (CjCDT), that induces cell cycle arrest and interleukin 8 (IL-8) secretion from intestinal epithelial cells in culture (2, 7, 13, 22). CDTs from other bacteria are also known to target immune cells and ultimately induce apoptosis (3, 19, 21). There have been two reports of apoptotic responses to C. jejuni (20, 24), and both suggested that mechanisms independent of CDT were responsible. Here, we confirm earlier observations of long-term campylobacter survival and replication within monocytes and the induction of apoptosis. Since CDTs of other pathogens induce apoptosis of lymphocytes (1, 3, 17, 19, 21), we reexamined the effect of CjCDT on human monocytes in culture.
Growth of C. jejuni in human 28SC monocyte cultures. The human monocyte line 28SC was infected with 81-176 at a multiplicity of infection (MOI) of 100:1 for 2 h. Extracellular bacteria were killed with 100 μg/ml gentamicin for an additional 2-h incubation. The cells were washed and reincubated, and bacterial counts were enumerated at various times. Peak levels of 81-176 were recovered from 48-hour cultures (Fig. 1), representing an approximate 3-log-unit increase in bacterial counts (from approximately 104 CFU/ml following gentamicin treatment to 107 CFU/ml after 48 h). Viable campylobacters were recovered up to day 7, consistent with the results of Kiehlbauch et al. (9). In contrast, in the absence of monocytes, 81-176 viability dropped rapidly, and no bacteria could be cultured after 72 h. Thus, replication of C. jejuni within monocytes was more extensive than the limited replication following internalization into epithelial cells (12).
Green fluorescent protein (GFP)-tagged 81-176 was imaged within 28SC cells using fluorescence microscopy. Generalized fluorescence (Fig. 2B) could be seen intracellularly within enlarged and irregularly shaped areas of cells (Fig. 2A). Using live/dead staining, the C. jejuni organisms appeared to be localized within vacuoles, either as single or multiple bacteria (Fig. 2C and D). The bacteria appeared motile within the vacuoles, consistent with expression of GFP under the control of the 28 promoter of the major flagellin (5).
Induction of chemokines from 28SC cells is independent of CDT. Viable campylobacters mediated chemokine responses from 28SC cells in 24-h cultures (Fig. 3A). 81-176 induced 1,689 ± 848 pg/ml IL-6 and 1,231 ± 479 pg/ml IL-8 in nine independent experiments. Since CDT is localized to bacterial membranes and is known to induce IL-8 secretion from intestinal epithelial cells (7), we compared the abilities of purified membrane proteins from DS105, a cdtA mutant deficient in CDT activity, and 81-176 to induce chemokine secretion from monocytes. When inoculated with 2 μg/ml bacterial proteins as described previously (6, 7), chemokine responses to 81-176 and DS105 proteins were similar (Fig. 3B). 81-176 and DS105 proteins induced 6,700 ± 2,409 and 3,324 ± 2,042 pg/ml IL-6 and 2,073 ± 467 and 1,909 ± 436 pg/ml IL-8, respectively. These data are consistent with previous reports implicating bacterial components, such as lipooligosaccharide, and not CjCDT in cytokine induction from monocytes (8).
Programmed cell death (PCD). Uninfected 28SC cell viability was >84% at 96 h (Fig. 4) and fell to 56.5% ± 0.7% by 168 h. In contrast, the viability of cells infected with 81-176 was <75% by 72 h and continued to decline to 7.2% ± 2.6% at 168 h. Annexin V binding to surface phosphatidylserine (PS) (15) was measured using the Guava Technologies Nexin assay (Guava Technologies, Hayward, CA). C. jejuni induced apoptosis of 28SC cells at levels significantly greater than that seen in uninoculated cultures (Fig. 5A). Only 9.5% ± 5.0% of cells in control cultures bound annexin V after 96 h, and this progressed to 24.4% ± 2.2% at 168 h. In contrast, by 48 h, 18.6% ± 2% of 81-176-infected monocytes were apoptotic. Maximal levels were evident at 144 h, when 55% ± 15% of the cells expressed surface PS.
CjCDT mediates apoptosis of 28SC cells. CjCDT has been shown to localize to membranes (6, 7). Membrane proteins (2 μg/ml) from 81-176, the cdtA mutant DS105, and its complement, DS105(pRAM18) (7), were added to 28SC cultures. Wild-type 81-176 membranes induced surface PS expression on 19.0% ± 2.3% of culture cells by 48 h (Fig. 5B) (P 0.001). Membranes of DS105 cdtA mediated PS expression on 9.0% ± 4% of the cells (P = 0.92). Membranes isolated from DS105(pRAM18), the CDT mutant complemented in trans, induced PS expression at levels comparable to those of the wild type.
To verify CjCDT-induced apoptosis by an alternate method, activation levels of pre- and postmitochondrial caspases following exposure to C. jejuni membranes were measured. In 8-h cultures, caspase 9, and to a lesser extent caspase 8, activities correlated with the presence of CjCDT (Fig. 6A and B). 81-176 membranes mediated 18,546 ± 1,486 luminescence units for caspase 9 (P 0.0001). Cultures inoculated with DS105 membranes induced 793 ± 1,759 units (P = 0.37). The highest activity was seen in cultures inoculated with DS105(pRAM18) membrane fractions (29,129 ± 2,477 units; P 0.0001). Similar results were observed for caspase 8 activity (Fig. 6B). These data indicate that caspases associated with intrinsic and external apoptosis pathways were activated in response to CjCDT (4), consistent with other CDTs (1, 3, 17, 19, 21).
Both previous reports of induction of apoptosis by C. jejuni in either chicken lymphocytes (24) or human monocytes (20) indicated that PCD was independent of CjCDT, although limited data were presented. Siegesmund et al. (20) reported that apoptosis of THP-1 monocytes required, instead, secretion of the Cia (campylobacter invasion antigen) proteins that are secreted through the flagellar secretion apparatus (10, 11, 18). We constructed site-specific insertional mutants of 81-176 defective in ciaB or flgB, a component of the flagellar basal body. Both mutants would be expected to be defective in secretion of the Cia proteins (10). However, both mutants induced levels of apoptosis comparable to those of 81-176 (data not shown).
C. jejuni can survive intracellularly in both intestinal epithelial cells and monocytes. The DNA damage induced by CjCDT appears to exert different effects on each cell type, which is likely a function of the p53 status of the line (1). In epithelial cells, CjCDT causes a G2/M block and induces high levels of chemokines. In contrast, release of proinflammatory chemokines from infected monocytes appears to be independent of CjCDT, and the cytotoxic effect is manifested by apoptosis. Survival within macrophages likely enhances the localized inflammatory response to C. jejuni and may also provide the bacteria with an immunologically privileged site and a mechanism for replication and dissemination within the host (12).
ACKNOWLEDGMENTS
We thank Cheryl Ewing and Anahita Kiavand for plasmid and mutant constructions, Shahida Baqar and Bob Lin for their expert advice on flow cytometry, and Chad Porter for help with statistical analysis.
This work was funded by the Military Infectious Diseases Program work unit no. 6000.RAD1.DA3.A0308.
REFERENCES
1. Cortes-Bratti, X., C. Karlsson, T. Lagergard, M. Thelestam, and T. Frisan. 2001. The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via DNA damage checkpoint pathways. Infect. Immun. 276:5296-5302.
2. Elwell, C. A., and L. A. Dreyfus. 2000. DNase I homologous residues in CdtB are critical for cytolethal distending toxin mediated cell cycle arrest. Mol. Microbiol. 37:952-963.
3. Gelfanova, V., E. J. Hansen, and S. M. Spinola. 1999. Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells. Infect. Immun. 67:6394-6402.
4. Green, D. R. 2000. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1-4.
5. Guerry, P., R. A. Alm, M. E. Power, S. M. Logan, and T. J. Trust. 1991. Role of two genes in Campylobacter motility. J. Bacteriol. 173:4757-4764.
6. Hickey, T. E., S. Baqar, A. L. Bourgeois, C. P. Ewing, and P. Guerry. 1999. Campylobacter jejuni stimulated secretion of interleukin-8 by INT407 cells. Infect. Immun. 67:88-93.
7. Hickey, T. E., A. L. McVeigh, D. A. Scott, R. E. Michietutti, A. Bixby, S. A. Carroll, A. L. Bourgeois, and P. Guerry. 2000. Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells. Infect. Immun. 68:6535-6541.
8. Jones, M. A., S. Totemeyer, D. J. Maskell, C. E. Bryant, and P. A. Barrow. 2003. Induction of proinflammatory responses in the human monocytic cell line THP-1 by Campylobacter jejuni. Infect. Immun. 71:2626-2633.
9. Kiehlbauch, J. A., R. A. Albach, L. L. Baum, and K. P. Chang. 1985. Phagocytosis of Campylobacter jejuni and its intracellular survival in mononuclear phagocytes. Infect. Immun. 48:446-451.
10. Konkel, M. E., B. J. Kim, V. Rivera-Amill, and S. G. Garvis. 1999. Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells. Mol. Microbiol. 32:691-701.
11. Konkel, M. E., J. D. Klena, V. Rivera-Amill, M. R. Monteville, D. Biswas, B. Raphael, and J. Mickelson. 2004. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J. Bacteriol. 186:3296-3303.
12. Kopecko, D. J., L. Hu, and K. J. M. Zaal. 2001. Campylobacter jejuni-microtubule-dependent invasion. Trends Microbiol. 9:389-396.
13. Lara-Tejero, M., and J. E. Galan. 2000. A bacterial toxin that controls cell cycle progression as a deoxyribonuclease-1 like protein. Science 290:354-357.
14. Larsen, J. C., C. Szymanski, and P. Guerry. 2004. N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176. J. Bacteriol. 186:6508-6514.
15. Martin, S. J., C. P. M. Reutelingsperger, A. J. McGahon, J. A. Rader, R. C. A. A. Van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182:1545-1556.
16. Miller, W. G., A. H. Bates, S. T. Horn, M. T. Brandl, M. R. Wachtel, and R. E. Mandrell. 2000. Detection on surfaces and in Caco-2 cells of Campylobacter jejuni transformed with new gfp, yfp, and cfp marker plasmids. Appl. Environ. Microbiol. 66:5426-5436.
17. Ohara, M., T. Hayashi, Y. Kusunoki, M. Miyauchi, T. Takata, and M. Sugai. 2004. Caspase-2 and caspase-7 are involved in cytolethal distending toxin-induced apoptosis in Jurkat and MOLT-4 T-cell lines. Infect. Immun. 72:871-879.
18. Rivera-Amill, V., B. J. Kim, J. Keshu, and M. E. Konkel. 2001. Secretion of the virulence associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal. J. Infect. Dis. 183:1607-1616.
19. Shenker, B. J., R. H. Hoffmaster, A. Zekavat, N. Yamaguchi, E. T. Lally, and D. Demuth. 2001. Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle. J. Immunol. 167:435-441.
20. Siegesmund, A. M., M. E. Konkel, J. D. Klena, and P. F. Mixter. 2004. Campylobacter jejuni infection of differentiated THP-1 macrophages results in interleukin 1 release and caspase-1-independent apoptosis. Microbiology 150:561-569.
21. Svensson, L. A., A. Tarkowski, M. Thelestam, and T. Lagergard. 2001. The impact of Hemophilus ducreyi cytolethal disetending toxin on cells involved in the immune response. Microb. Pathog. 30:157-166.
22. Whitehouse, C. A., P. B. Balbo, E. C. Pesci, D. L. Cottle, P. M. Mirabito, and C. L. Pickett. 1998. Campylobacter jejuni cytolethal distending toxin causes a G2 phase cell cycle block. Infect. Immun. 66:1934-1940.
23. Yao, R., R. A. Alm, T. J. Trust, and P. Guerry. 1993. Construction of new Campylobacter cloning vectors and a new chloramphenicol mutational cassette. Gene 130:127-130.
24. Zhu, J., R. J. Meinersmann, K. L. Hiett, and D. L. Evans. 1999. Apoptotic effect of outer-membrane proteins from Campylobacter jejuni on chicken lymphocytes. Curr. Microbiol. 38:244-249.(Thomas E. Hickey, Gary Ma)
ABSTRACT
Campylobacter jejuni 81-176 is capable of extensive replication within human monocytic cell vacuoles and induces apoptotic death via cytolethal distending toxin.
TEXT
Our understanding of the interaction of Campylobacter jejuni with immune effector cells is limited. C. jejuni can survive phagocytosis by human mononuclear cells and remain viable for extended periods (9), induce secretion of chemokines (8), and mediate apoptosis (20). C. jejuni elaborates a well-characterized toxin, cytolethal distending toxin (CjCDT), that induces cell cycle arrest and interleukin 8 (IL-8) secretion from intestinal epithelial cells in culture (2, 7, 13, 22). CDTs from other bacteria are also known to target immune cells and ultimately induce apoptosis (3, 19, 21). There have been two reports of apoptotic responses to C. jejuni (20, 24), and both suggested that mechanisms independent of CDT were responsible. Here, we confirm earlier observations of long-term campylobacter survival and replication within monocytes and the induction of apoptosis. Since CDTs of other pathogens induce apoptosis of lymphocytes (1, 3, 17, 19, 21), we reexamined the effect of CjCDT on human monocytes in culture.
Growth of C. jejuni in human 28SC monocyte cultures. The human monocyte line 28SC was infected with 81-176 at a multiplicity of infection (MOI) of 100:1 for 2 h. Extracellular bacteria were killed with 100 μg/ml gentamicin for an additional 2-h incubation. The cells were washed and reincubated, and bacterial counts were enumerated at various times. Peak levels of 81-176 were recovered from 48-hour cultures (Fig. 1), representing an approximate 3-log-unit increase in bacterial counts (from approximately 104 CFU/ml following gentamicin treatment to 107 CFU/ml after 48 h). Viable campylobacters were recovered up to day 7, consistent with the results of Kiehlbauch et al. (9). In contrast, in the absence of monocytes, 81-176 viability dropped rapidly, and no bacteria could be cultured after 72 h. Thus, replication of C. jejuni within monocytes was more extensive than the limited replication following internalization into epithelial cells (12).
Green fluorescent protein (GFP)-tagged 81-176 was imaged within 28SC cells using fluorescence microscopy. Generalized fluorescence (Fig. 2B) could be seen intracellularly within enlarged and irregularly shaped areas of cells (Fig. 2A). Using live/dead staining, the C. jejuni organisms appeared to be localized within vacuoles, either as single or multiple bacteria (Fig. 2C and D). The bacteria appeared motile within the vacuoles, consistent with expression of GFP under the control of the 28 promoter of the major flagellin (5).
Induction of chemokines from 28SC cells is independent of CDT. Viable campylobacters mediated chemokine responses from 28SC cells in 24-h cultures (Fig. 3A). 81-176 induced 1,689 ± 848 pg/ml IL-6 and 1,231 ± 479 pg/ml IL-8 in nine independent experiments. Since CDT is localized to bacterial membranes and is known to induce IL-8 secretion from intestinal epithelial cells (7), we compared the abilities of purified membrane proteins from DS105, a cdtA mutant deficient in CDT activity, and 81-176 to induce chemokine secretion from monocytes. When inoculated with 2 μg/ml bacterial proteins as described previously (6, 7), chemokine responses to 81-176 and DS105 proteins were similar (Fig. 3B). 81-176 and DS105 proteins induced 6,700 ± 2,409 and 3,324 ± 2,042 pg/ml IL-6 and 2,073 ± 467 and 1,909 ± 436 pg/ml IL-8, respectively. These data are consistent with previous reports implicating bacterial components, such as lipooligosaccharide, and not CjCDT in cytokine induction from monocytes (8).
Programmed cell death (PCD). Uninfected 28SC cell viability was >84% at 96 h (Fig. 4) and fell to 56.5% ± 0.7% by 168 h. In contrast, the viability of cells infected with 81-176 was <75% by 72 h and continued to decline to 7.2% ± 2.6% at 168 h. Annexin V binding to surface phosphatidylserine (PS) (15) was measured using the Guava Technologies Nexin assay (Guava Technologies, Hayward, CA). C. jejuni induced apoptosis of 28SC cells at levels significantly greater than that seen in uninoculated cultures (Fig. 5A). Only 9.5% ± 5.0% of cells in control cultures bound annexin V after 96 h, and this progressed to 24.4% ± 2.2% at 168 h. In contrast, by 48 h, 18.6% ± 2% of 81-176-infected monocytes were apoptotic. Maximal levels were evident at 144 h, when 55% ± 15% of the cells expressed surface PS.
CjCDT mediates apoptosis of 28SC cells. CjCDT has been shown to localize to membranes (6, 7). Membrane proteins (2 μg/ml) from 81-176, the cdtA mutant DS105, and its complement, DS105(pRAM18) (7), were added to 28SC cultures. Wild-type 81-176 membranes induced surface PS expression on 19.0% ± 2.3% of culture cells by 48 h (Fig. 5B) (P 0.001). Membranes of DS105 cdtA mediated PS expression on 9.0% ± 4% of the cells (P = 0.92). Membranes isolated from DS105(pRAM18), the CDT mutant complemented in trans, induced PS expression at levels comparable to those of the wild type.
To verify CjCDT-induced apoptosis by an alternate method, activation levels of pre- and postmitochondrial caspases following exposure to C. jejuni membranes were measured. In 8-h cultures, caspase 9, and to a lesser extent caspase 8, activities correlated with the presence of CjCDT (Fig. 6A and B). 81-176 membranes mediated 18,546 ± 1,486 luminescence units for caspase 9 (P 0.0001). Cultures inoculated with DS105 membranes induced 793 ± 1,759 units (P = 0.37). The highest activity was seen in cultures inoculated with DS105(pRAM18) membrane fractions (29,129 ± 2,477 units; P 0.0001). Similar results were observed for caspase 8 activity (Fig. 6B). These data indicate that caspases associated with intrinsic and external apoptosis pathways were activated in response to CjCDT (4), consistent with other CDTs (1, 3, 17, 19, 21).
Both previous reports of induction of apoptosis by C. jejuni in either chicken lymphocytes (24) or human monocytes (20) indicated that PCD was independent of CjCDT, although limited data were presented. Siegesmund et al. (20) reported that apoptosis of THP-1 monocytes required, instead, secretion of the Cia (campylobacter invasion antigen) proteins that are secreted through the flagellar secretion apparatus (10, 11, 18). We constructed site-specific insertional mutants of 81-176 defective in ciaB or flgB, a component of the flagellar basal body. Both mutants would be expected to be defective in secretion of the Cia proteins (10). However, both mutants induced levels of apoptosis comparable to those of 81-176 (data not shown).
C. jejuni can survive intracellularly in both intestinal epithelial cells and monocytes. The DNA damage induced by CjCDT appears to exert different effects on each cell type, which is likely a function of the p53 status of the line (1). In epithelial cells, CjCDT causes a G2/M block and induces high levels of chemokines. In contrast, release of proinflammatory chemokines from infected monocytes appears to be independent of CjCDT, and the cytotoxic effect is manifested by apoptosis. Survival within macrophages likely enhances the localized inflammatory response to C. jejuni and may also provide the bacteria with an immunologically privileged site and a mechanism for replication and dissemination within the host (12).
ACKNOWLEDGMENTS
We thank Cheryl Ewing and Anahita Kiavand for plasmid and mutant constructions, Shahida Baqar and Bob Lin for their expert advice on flow cytometry, and Chad Porter for help with statistical analysis.
This work was funded by the Military Infectious Diseases Program work unit no. 6000.RAD1.DA3.A0308.
REFERENCES
1. Cortes-Bratti, X., C. Karlsson, T. Lagergard, M. Thelestam, and T. Frisan. 2001. The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via DNA damage checkpoint pathways. Infect. Immun. 276:5296-5302.
2. Elwell, C. A., and L. A. Dreyfus. 2000. DNase I homologous residues in CdtB are critical for cytolethal distending toxin mediated cell cycle arrest. Mol. Microbiol. 37:952-963.
3. Gelfanova, V., E. J. Hansen, and S. M. Spinola. 1999. Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells. Infect. Immun. 67:6394-6402.
4. Green, D. R. 2000. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1-4.
5. Guerry, P., R. A. Alm, M. E. Power, S. M. Logan, and T. J. Trust. 1991. Role of two genes in Campylobacter motility. J. Bacteriol. 173:4757-4764.
6. Hickey, T. E., S. Baqar, A. L. Bourgeois, C. P. Ewing, and P. Guerry. 1999. Campylobacter jejuni stimulated secretion of interleukin-8 by INT407 cells. Infect. Immun. 67:88-93.
7. Hickey, T. E., A. L. McVeigh, D. A. Scott, R. E. Michietutti, A. Bixby, S. A. Carroll, A. L. Bourgeois, and P. Guerry. 2000. Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells. Infect. Immun. 68:6535-6541.
8. Jones, M. A., S. Totemeyer, D. J. Maskell, C. E. Bryant, and P. A. Barrow. 2003. Induction of proinflammatory responses in the human monocytic cell line THP-1 by Campylobacter jejuni. Infect. Immun. 71:2626-2633.
9. Kiehlbauch, J. A., R. A. Albach, L. L. Baum, and K. P. Chang. 1985. Phagocytosis of Campylobacter jejuni and its intracellular survival in mononuclear phagocytes. Infect. Immun. 48:446-451.
10. Konkel, M. E., B. J. Kim, V. Rivera-Amill, and S. G. Garvis. 1999. Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells. Mol. Microbiol. 32:691-701.
11. Konkel, M. E., J. D. Klena, V. Rivera-Amill, M. R. Monteville, D. Biswas, B. Raphael, and J. Mickelson. 2004. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J. Bacteriol. 186:3296-3303.
12. Kopecko, D. J., L. Hu, and K. J. M. Zaal. 2001. Campylobacter jejuni-microtubule-dependent invasion. Trends Microbiol. 9:389-396.
13. Lara-Tejero, M., and J. E. Galan. 2000. A bacterial toxin that controls cell cycle progression as a deoxyribonuclease-1 like protein. Science 290:354-357.
14. Larsen, J. C., C. Szymanski, and P. Guerry. 2004. N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176. J. Bacteriol. 186:6508-6514.
15. Martin, S. J., C. P. M. Reutelingsperger, A. J. McGahon, J. A. Rader, R. C. A. A. Van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182:1545-1556.
16. Miller, W. G., A. H. Bates, S. T. Horn, M. T. Brandl, M. R. Wachtel, and R. E. Mandrell. 2000. Detection on surfaces and in Caco-2 cells of Campylobacter jejuni transformed with new gfp, yfp, and cfp marker plasmids. Appl. Environ. Microbiol. 66:5426-5436.
17. Ohara, M., T. Hayashi, Y. Kusunoki, M. Miyauchi, T. Takata, and M. Sugai. 2004. Caspase-2 and caspase-7 are involved in cytolethal distending toxin-induced apoptosis in Jurkat and MOLT-4 T-cell lines. Infect. Immun. 72:871-879.
18. Rivera-Amill, V., B. J. Kim, J. Keshu, and M. E. Konkel. 2001. Secretion of the virulence associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal. J. Infect. Dis. 183:1607-1616.
19. Shenker, B. J., R. H. Hoffmaster, A. Zekavat, N. Yamaguchi, E. T. Lally, and D. Demuth. 2001. Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle. J. Immunol. 167:435-441.
20. Siegesmund, A. M., M. E. Konkel, J. D. Klena, and P. F. Mixter. 2004. Campylobacter jejuni infection of differentiated THP-1 macrophages results in interleukin 1 release and caspase-1-independent apoptosis. Microbiology 150:561-569.
21. Svensson, L. A., A. Tarkowski, M. Thelestam, and T. Lagergard. 2001. The impact of Hemophilus ducreyi cytolethal disetending toxin on cells involved in the immune response. Microb. Pathog. 30:157-166.
22. Whitehouse, C. A., P. B. Balbo, E. C. Pesci, D. L. Cottle, P. M. Mirabito, and C. L. Pickett. 1998. Campylobacter jejuni cytolethal distending toxin causes a G2 phase cell cycle block. Infect. Immun. 66:1934-1940.
23. Yao, R., R. A. Alm, T. J. Trust, and P. Guerry. 1993. Construction of new Campylobacter cloning vectors and a new chloramphenicol mutational cassette. Gene 130:127-130.
24. Zhu, J., R. J. Meinersmann, K. L. Hiett, and D. L. Evans. 1999. Apoptotic effect of outer-membrane proteins from Campylobacter jejuni on chicken lymphocytes. Curr. Microbiol. 38:244-249.(Thomas E. Hickey, Gary Ma)