Accepted Article
Received Date : 21-Jul-2014 Accepted Date : 21-Jul-2014 Article type : Commentary Editor: Peter Timms
Question the questions on CPAF
Guangming Zhong Department of Microbiology and Immunology University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive, San Antonio, TX78229 Phone: 210-567-1169; Email: [email protected]
This is in response to the editorial commentary on CPAF substrates and secretion authored by Drs. Patrick Bavoil and Gerald Byrne (1).
Chlamydial protease-like activity factor (CPAF) was first purified in two fragments (2) that formed intramolecular heterodimers (3, 4) for acquiring a serine protease activity (5-7). The crystal structure revealed a proximity-dependent super-dimerization and sequential processing mechanism for CPAF activation (7). CPAF was characterized as a secdependent secretion protein (8) that localized to the host cytosol (2, 9-13) but without colocalization with the secreted Pgp3 protein (14, 15) and implicated functionally in the interaction of a number of host cell substrate targets important to chlamydial pathogenesis (16). However, in 2012 it was unambiguously shown that many of CPAF’s previously identified host substrates were the result of an imprecise method that technically failed to inactive the protease (17). This report triggered controversy and discussions on the authenticity of CPAF’s function (18, 19). The recent generation of CPAF-deficient chlamydial
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/2049-632X.12205 This article is protected by copyright. All rights reserved.
Accepted Article
organisms has provided a genetic basis for investigating the functionality of CPAF (20). This
1.
2.
3.
study, based on a host cytoskeletal protein target that was affected only very late in the chlamydial growth cycle in some infected cells with lysis of the inclusion membrane, raised the question on whether CPAF is a bona fide secreted protein. Based primarily on this observation, Bavoil and Byrne took the liberty to write the aforementioned commentary that further raised the question on the authenticity of CPAF secretion. They claim that the only experimental evidence supporting CPAF secretion into the cytosol is by immunofluorescent staining that required sample processing and fixation conditions that could result in a passive leaking of CPAF into host cell cytosol. This conclusion, in the absence of any experimental findings by the authors, is too premature and academically risky as it may cause unmerited confusion among investigators; especially those who are new to the chlamydial research field and are most impressionable by the Bavoil and Byrne commentary.
CPAF is a
conserved protease common to all Chlamydiaceae (21) whose secretion and cytosolic location needs further investigation to definitively elucidate its function in chlamydial pathogenesis. The optimistic viewpoint of this rebuttal is that with the current advances in chlamydial genetics we now have tools to conduct experiments that will define CPAF’s function (22-27), not simply speculate on it, and will resolve this question unequivocally.
References Bavoil, P. M., and G. I. Byrne. 2014. Analysis of CPAF mutants: new functions, new questions (The ins and outs of a chlamydial protease). Pathog Dis Jun 19: 10.1111/20491632X.12194. Zhong, G., P. Fan, H. Ji, F. Dong, and Y. Huang. 2001. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193: 935-942. Dong, F., M. Pirbhai, Y. Zhong, and G. Zhong. 2004. Cleavage-dependent activation of a chlamydia-secreted protease. Molecular microbiology 52: 1487-1494.
This article is protected by copyright. All rights reserved.
Accepted Article
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Dong, F., J. Sharma, Y. Xiao, Y. Zhong, and G. Zhong. 2004. Intramolecular dimerization is required for the chlamydia-secreted protease CPAF to degrade host transcriptional factors. Infection and immunity 72: 3869-3875. Chen, D., J. Chai, P. J. Hart, and G. Zhong. 2009. Identifying catalytic residues in CPAF, a Chlamydia-secreted protease. Arch Biochem Biophys 485: 16-23. Chen, D., L. Lei, R. Flores, Z. Huang, Z. Wu, J. Chai, and G. Zhong. 2010. Autoprocessing and self-activation of the secreted protease CPAF in Chlamydia-infected cells. Microb Pathog 49: 164-173. Huang, Z., Y. Feng, D. Chen, X. Wu, S. Huang, X. Wang, X. Xiao, W. Li, N. Huang, L. Gu, G. Zhong, and J. Chai. 2008. Structural Basis for Activation and Inhibition of the Secreted Chlamydia Protease CPAF. Cell Host Microbe 4: 529-542. Chen, D., L. Lei, C. Lu, R. Flores, M. P. DeLisa, T. C. Roberts, F. E. Romesberg, and G. Zhong. 2010. Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway. Microbiology 156: 3031-3040. Kleba, B., and R. S. Stephens. 2008. Chlamydial effector proteins localized to the host cell cytoplasmic compartment. Infection and immunity 76: 4842-4850. Wang, J., K. M. Frohlich, L. Buckner, A. J. Quayle, M. Luo, X. Feng, W. Beatty, Z. Hua, X. Rao, M. E. Lewis, K. Sorrells, K. Santiago, G. Zhong, and L. Shen. 2011. Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells. Microbiology 157: 2759-2771. Heuer, D., V. Brinkmann, T. F. Meyer, and A. J. Szczepek. 2003. Expression and translocation of chlamydial protease during acute and persistent infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia) pneumoniae. Cell Microbiol 5: 315-322. Bauler, L. D., and T. Hackstadt. 2014. Expression and targeting of secreted proteins from Chlamydia trachomatis. Journal of bacteriology 196: 1325-1334. Fan, P., F. Dong, Y. Huang, and G. Zhong. 2002. Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors required for
This article is protected by copyright. All rights reserved.
Accepted Article
[correction of factors is required for] major histocompatibility complex antigen expression.
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Infection and immunity 70: 345-349. Li, Z., D. Chen, Y. Zhong, S. Wang, and G. Zhong. 2008. The chlamydial plasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells. Infection and immunity 76: 3415-3428. Chen, D., L. Lei, C. Lu, A. Galaleldeen, P. J. Hart, and G. Zhong. 2010. Characterization of Pgp3, a Chlamydia trachomatis plasmid-encoded immunodominant antigen. Journal of bacteriology 192: 6017-6024. Zhong, G. 2011. Chlamydia trachomatis secretion of proteases for manipulating host signaling pathways. Front Microbiol 2: 14-19. Chen, A. L., K. A. Johnson, J. K. Lee, C. Sütterlin, and M. Tan. 2012. CPAF: a Chlamydial protease in search of an authentic substrate. PLoS Pathog 8: e1002842. Conrad, T. A., Z. Yang, D. Ojcius, and G. Zhong. 2013. A path forward for the chlamydial virulence factor CPAF. Microbes Infect 15: 1026-1032. Hacker, G. 2014. The chlamydial protease CPAF: important or not, important for what? Microbes Infect 16: 367-370. Snavely, E. A., M. Kokes, J. D. Dunn, H. A. Saka, B. D. Nguyen, R. J. Bastidas, D. G. McCafferty, and R. H. Valdivia. 2014. Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis May 16: doi: 10.1111/2049-1632X.12179. . Dong, F., Y. Zhong, B. Arulanandam, and G. Zhong. 2005. Production of a proteolytically active protein, chlamydial protease/proteasome-like activity factor, by five different Chlamydia species. Infection and immunity 73: 1868-1872. Wang, Y., S. Kahane, L. T. Cutcliffe, R. J. Skilton, P. R. Lambden, and I. N. Clarke. 2011. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7: e1002258. Song, L., J. H. Carlson, W. M. Whitmire, L. Kari, K. Virtaneva, D. E. Sturdevant, H. Watkins, B. Zhou, G. L. Sturdevant, S. F. Porcella, G. McClarty, and H. D. Caldwell. 2013. Chlamydia
This article is protected by copyright. All rights reserved.
Accepted Article
trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence-associated
24.
25.
26.
27.
genes. Infection and immunity 81: 636-644. Gong, S., Z. Yang, L. Lei, L. Shen, and G. Zhong. 2013. Characterization of Chlamydia trachomatis Plasmid-Encoded Open Reading Frames. Journal of bacteriology 195: 38193826. Ding, H., S. Gong, Y. Tian, Z. Yang, R. Brunham, and G. Zhong. 2013. Transformation of sexually transmitted infection-causing serovars of Chlamydia trachomatis using blasticidin for selection PloS one 8: e80534. Liu, Y., C. Chen, S. Gong, S. Hou, M. Qi, Q. Liu, J. Baseman, and G. Zhong. 2014. Transformation of Chlamydia muridarum reveals a role for Pgp5 in suppression of plasmiddependent gene expression. Journal of bacteriology 196: 989-998. Johnson, C. M., and D. J. Fisher. 2013. Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron. PloS one 8: e83989.
This article is protected by copyright. All rights reserved.
Received Date : 21-Jul-2014 Accepted Date : 21-Jul-2014 Article type : Commentary Editor: Peter Timms
Question the questions on CPAF
Guangming Zhong Department of Microbiology and Immunology University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive, San Antonio, TX78229 Phone: 210-567-1169; Email: [email protected]
This is in response to the editorial commentary on CPAF substrates and secretion authored by Drs. Patrick Bavoil and Gerald Byrne (1).
Chlamydial protease-like activity factor (CPAF) was first purified in two fragments (2) that formed intramolecular heterodimers (3, 4) for acquiring a serine protease activity (5-7). The crystal structure revealed a proximity-dependent super-dimerization and sequential processing mechanism for CPAF activation (7). CPAF was characterized as a secdependent secretion protein (8) that localized to the host cytosol (2, 9-13) but without colocalization with the secreted Pgp3 protein (14, 15) and implicated functionally in the interaction of a number of host cell substrate targets important to chlamydial pathogenesis (16). However, in 2012 it was unambiguously shown that many of CPAF’s previously identified host substrates were the result of an imprecise method that technically failed to inactive the protease (17). This report triggered controversy and discussions on the authenticity of CPAF’s function (18, 19). The recent generation of CPAF-deficient chlamydial
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/2049-632X.12205 This article is protected by copyright. All rights reserved.
Accepted Article
organisms has provided a genetic basis for investigating the functionality of CPAF (20). This
1.
2.
3.
study, based on a host cytoskeletal protein target that was affected only very late in the chlamydial growth cycle in some infected cells with lysis of the inclusion membrane, raised the question on whether CPAF is a bona fide secreted protein. Based primarily on this observation, Bavoil and Byrne took the liberty to write the aforementioned commentary that further raised the question on the authenticity of CPAF secretion. They claim that the only experimental evidence supporting CPAF secretion into the cytosol is by immunofluorescent staining that required sample processing and fixation conditions that could result in a passive leaking of CPAF into host cell cytosol. This conclusion, in the absence of any experimental findings by the authors, is too premature and academically risky as it may cause unmerited confusion among investigators; especially those who are new to the chlamydial research field and are most impressionable by the Bavoil and Byrne commentary.
CPAF is a
conserved protease common to all Chlamydiaceae (21) whose secretion and cytosolic location needs further investigation to definitively elucidate its function in chlamydial pathogenesis. The optimistic viewpoint of this rebuttal is that with the current advances in chlamydial genetics we now have tools to conduct experiments that will define CPAF’s function (22-27), not simply speculate on it, and will resolve this question unequivocally.
References Bavoil, P. M., and G. I. Byrne. 2014. Analysis of CPAF mutants: new functions, new questions (The ins and outs of a chlamydial protease). Pathog Dis Jun 19: 10.1111/20491632X.12194. Zhong, G., P. Fan, H. Ji, F. Dong, and Y. Huang. 2001. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193: 935-942. Dong, F., M. Pirbhai, Y. Zhong, and G. Zhong. 2004. Cleavage-dependent activation of a chlamydia-secreted protease. Molecular microbiology 52: 1487-1494.
This article is protected by copyright. All rights reserved.
Accepted Article
4.
5.
6.
7.
8.
9.
10.
11.
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Dong, F., J. Sharma, Y. Xiao, Y. Zhong, and G. Zhong. 2004. Intramolecular dimerization is required for the chlamydia-secreted protease CPAF to degrade host transcriptional factors. Infection and immunity 72: 3869-3875. Chen, D., J. Chai, P. J. Hart, and G. Zhong. 2009. Identifying catalytic residues in CPAF, a Chlamydia-secreted protease. Arch Biochem Biophys 485: 16-23. Chen, D., L. Lei, R. Flores, Z. Huang, Z. Wu, J. Chai, and G. Zhong. 2010. Autoprocessing and self-activation of the secreted protease CPAF in Chlamydia-infected cells. Microb Pathog 49: 164-173. Huang, Z., Y. Feng, D. Chen, X. Wu, S. Huang, X. Wang, X. Xiao, W. Li, N. Huang, L. Gu, G. Zhong, and J. Chai. 2008. Structural Basis for Activation and Inhibition of the Secreted Chlamydia Protease CPAF. Cell Host Microbe 4: 529-542. Chen, D., L. Lei, C. Lu, R. Flores, M. P. DeLisa, T. C. Roberts, F. E. Romesberg, and G. Zhong. 2010. Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway. Microbiology 156: 3031-3040. Kleba, B., and R. S. Stephens. 2008. Chlamydial effector proteins localized to the host cell cytoplasmic compartment. Infection and immunity 76: 4842-4850. Wang, J., K. M. Frohlich, L. Buckner, A. J. Quayle, M. Luo, X. Feng, W. Beatty, Z. Hua, X. Rao, M. E. Lewis, K. Sorrells, K. Santiago, G. Zhong, and L. Shen. 2011. Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells. Microbiology 157: 2759-2771. Heuer, D., V. Brinkmann, T. F. Meyer, and A. J. Szczepek. 2003. Expression and translocation of chlamydial protease during acute and persistent infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia) pneumoniae. Cell Microbiol 5: 315-322. Bauler, L. D., and T. Hackstadt. 2014. Expression and targeting of secreted proteins from Chlamydia trachomatis. Journal of bacteriology 196: 1325-1334. Fan, P., F. Dong, Y. Huang, and G. Zhong. 2002. Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors required for
This article is protected by copyright. All rights reserved.
Accepted Article
[correction of factors is required for] major histocompatibility complex antigen expression.
14.
15.
16.
17.
18.
19.
20.
21.
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Infection and immunity 70: 345-349. Li, Z., D. Chen, Y. Zhong, S. Wang, and G. Zhong. 2008. The chlamydial plasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells. Infection and immunity 76: 3415-3428. Chen, D., L. Lei, C. Lu, A. Galaleldeen, P. J. Hart, and G. Zhong. 2010. Characterization of Pgp3, a Chlamydia trachomatis plasmid-encoded immunodominant antigen. Journal of bacteriology 192: 6017-6024. Zhong, G. 2011. Chlamydia trachomatis secretion of proteases for manipulating host signaling pathways. Front Microbiol 2: 14-19. Chen, A. L., K. A. Johnson, J. K. Lee, C. Sütterlin, and M. Tan. 2012. CPAF: a Chlamydial protease in search of an authentic substrate. PLoS Pathog 8: e1002842. Conrad, T. A., Z. Yang, D. Ojcius, and G. Zhong. 2013. A path forward for the chlamydial virulence factor CPAF. Microbes Infect 15: 1026-1032. Hacker, G. 2014. The chlamydial protease CPAF: important or not, important for what? Microbes Infect 16: 367-370. Snavely, E. A., M. Kokes, J. D. Dunn, H. A. Saka, B. D. Nguyen, R. J. Bastidas, D. G. McCafferty, and R. H. Valdivia. 2014. Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis May 16: doi: 10.1111/2049-1632X.12179. . Dong, F., Y. Zhong, B. Arulanandam, and G. Zhong. 2005. Production of a proteolytically active protein, chlamydial protease/proteasome-like activity factor, by five different Chlamydia species. Infection and immunity 73: 1868-1872. Wang, Y., S. Kahane, L. T. Cutcliffe, R. J. Skilton, P. R. Lambden, and I. N. Clarke. 2011. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7: e1002258. Song, L., J. H. Carlson, W. M. Whitmire, L. Kari, K. Virtaneva, D. E. Sturdevant, H. Watkins, B. Zhou, G. L. Sturdevant, S. F. Porcella, G. McClarty, and H. D. Caldwell. 2013. Chlamydia
This article is protected by copyright. All rights reserved.
Accepted Article
trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence-associated
24.
25.
26.
27.
genes. Infection and immunity 81: 636-644. Gong, S., Z. Yang, L. Lei, L. Shen, and G. Zhong. 2013. Characterization of Chlamydia trachomatis Plasmid-Encoded Open Reading Frames. Journal of bacteriology 195: 38193826. Ding, H., S. Gong, Y. Tian, Z. Yang, R. Brunham, and G. Zhong. 2013. Transformation of sexually transmitted infection-causing serovars of Chlamydia trachomatis using blasticidin for selection PloS one 8: e80534. Liu, Y., C. Chen, S. Gong, S. Hou, M. Qi, Q. Liu, J. Baseman, and G. Zhong. 2014. Transformation of Chlamydia muridarum reveals a role for Pgp5 in suppression of plasmiddependent gene expression. Journal of bacteriology 196: 989-998. Johnson, C. M., and D. J. Fisher. 2013. Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron. PloS one 8: e83989.
This article is protected by copyright. All rights reserved.
