Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth


Bacterial virulence determinants can be identified, according to the molecular Koch's postulates1, if inactivation of a gene associated with a suspected virulence trait results in a loss in pathogenicity. This approach is commonly used with genetically tractable organisms. However, the current lack of tools for targeted gene disruptions in obligate intracellular microbial pathogens seriously hampers the identification of their virulence factors. Here we demonstrate an approach to studying potential virulence factors of genetically intractable organisms, such as Chlamydia. Heterologous expression of Chlamydia pneumoniae CopN in yeast and mammalian cells resulted in a cell cycle arrest, presumably owing to alterations in the microtubule cytoskeleton. A screen of a small molecule library identified two compounds that alleviated CopN-induced growth inhibition in yeast. These compounds interfered with C. pneumoniae replication in mammalian cells, presumably by ‘knocking out’ CopN function, revealing an essential role of CopN in the support of C. pneumoniae growth during infection. This work demonstrates the role of a specific chlamydial protein in virulence. The chemical biology approach described here can be used to identify virulence factors, and the reverse chemical genetic strategy can result in the identification of lead compounds for the development of novel therapeutics.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: CopN expression inhibits yeast growth and results in the accumulation of large-budded yeast.
Figure 2: CopN expression induces a cell cycle arrest in both yeast and mammalian cells due to disruption of microtubules.
Figure 3: The small molecule inhibitors 0433YC1 and 0433YC2 alleviate yeast growth inhibition due to CopN expression.
Figure 4: The CopN inhibitors 0433YC1 and 0433YC2 inhibit C. pneumoniae replication in host cells.


  1. Falkow, S. Molecular Koch's postulates applied to microbial pathogenicity. Rev. Infect. Dis. 10 (Suppl. 2). S274–S276 (1988)

    Article  Google Scholar 

  2. Campbell, L. A. & Kuo, C. C. Chlamydia pneumoniae — an infectious risk factor for atherosclerosis? Nature Rev. Microbiol. 2, 23–32 (2004)

    CAS  Article  Google Scholar 

  3. Hackstadt, T. in Chlamydia: Intracellular Biology, Pathogenesis, and Immunity (ed. Stephens, R. S.) 101–138 (ASM Press, 1999)

    Book  Google Scholar 

  4. Crocker, T. T., Pelc, S. R., Nielsen, B. I., Eastwood, J. M. & Banks, J. Population dynamics and deoxyribonucleic acid synthesis in HeLa cells infected with an ornithosis agent. J. Infect. Dis. 115, 105–122 (1965)

    CAS  Article  Google Scholar 

  5. Hackstadt, T., Scidmore, M. A. & Rockey, D. D. Lipid metabolism in Chlamydia trachomatis-infected cells: Directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc. Natl Acad. Sci. USA 92, 4877–4881 (1995)

    ADS  CAS  Article  Google Scholar 

  6. Fan, T. et al. Inhibition of apoptosis in Chlamydia-infected cells: Blockade of mitochondrial cytochrome c release and caspase activation. J. Exp. Med. 187, 487–496 (1998)

    CAS  Article  Google Scholar 

  7. Carabeo, R. A., Grieshaber, S. S., Fischer, E. & Hackstadt, T. Chlamydia trachomatis induces remodeling of the actin cytoskeleton during attachment and entry into HeLa cells. Infect. Immun. 70, 3793–3803 (2002)

    CAS  Article  Google Scholar 

  8. Carabeo, R. A., Mead, D. J. & Hackstadt, T. Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc. Natl Acad. Sci. USA 100, 6771–6776 (2003)

    ADS  CAS  Article  Google Scholar 

  9. Su, H. et al. Activation of Raf/MEK/ERK/cPLA2 signaling pathway is essential for chlamydial acquisition of host glycerophospholipids. J. Biol. Chem. 279, 9409–9416 (2004)

    CAS  Article  Google Scholar 

  10. Hsia, R. C., Pannekoek, Y., Ingerowski, E. & Bavoil, P. M. Type III secretion genes identify a putative virulence locus of Chlamydia . Mol. Microbiol. 25, 351–359 (1997)

    CAS  Article  Google Scholar 

  11. Fields, K. A., Mead, D. J., Dooley, C. A. & Hackstadt, T. Chlamydia trachomatis type III secretion: Evidence for a functional apparatus during early-cycle development. Mol. Microbiol. 48, 671–683 (2003)

    CAS  Article  Google Scholar 

  12. Peters, J., Wilson, D. P., Myers, G., Timms, P. & Bavoil, P. M. Type III secretion a la Chlamydia . Trends Microbiol. 15, 241–251 (2007)

    CAS  Article  Google Scholar 

  13. Valdivia, R. H. Chlamydia effector proteins and new insights into chlamydial cellular microbiology. Curr. Opin. Microbiol. 11, 53–59 (2008)

    CAS  Article  Google Scholar 

  14. Lesser, C. F. & Miller, S. I. Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection. EMBO J. 20, 1840–1849 (2001)

    CAS  Article  Google Scholar 

  15. Siggers, K. A. & Lesser, C. F. The yeast Saccharomyces cerevisiae: A versatile model system for the identification and characterization of bacterial virulence proteins. Cell Host Microbe 4, 8–15 (2008)

    CAS  Article  Google Scholar 

  16. Lugert, R., Kuhns, M., Polch, T. & Gross, U. Expression and localization of type III secretion-related proteins of Chlamydia pneumoniae . Med. Microbiol. Immunol. (Berl.) 193, 163–171 (2004)

    CAS  Article  Google Scholar 

  17. Verma, A. & Maurelli, A. T. Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1. Infect. Immun. 71, 5772–5784 (2003)

    CAS  Article  Google Scholar 

  18. Li, D. et al. High-yield culture and purification of Chlamydiaceae bacteria. J. Microbiol. Methods 61, 17–24 (2005)

    CAS  Article  Google Scholar 

  19. Khan, M. A., Potter, C. W. & Sharrard, R. M. A reverse transcriptase-PCR based assay for in-vitro antibiotic susceptibility testing of Chlamydia pneumoniae . J. Antimicrob. Chemother. 37, 677–685 (1996)

    CAS  Article  Google Scholar 

  20. Cross, N. A. et al. Antimicrobial susceptibility testing of Chlamydia trachomatis using a reverse transcriptase PCR-based method. Antimicrob. Agents Chemother. 43, 2311–2313 (1999)

    CAS  Article  Google Scholar 

  21. Huang, J. et al. The quantity of nitric oxide released by macrophages regulates Chlamydia-induced disease. Proc. Natl Acad. Sci. USA 99, 3914–3919 (2002)

    ADS  CAS  Article  Google Scholar 

  22. Moulder, J. W. The relation of basic biology to pathogenic potential in the genus Chlamydia . Infection 10 (Suppl 1). S10–S18 (1982)

    Article  Google Scholar 

  23. Sisko, J. L., Spaeth, K., Kumar, Y. & Valdivia, R. H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system. Mol. Microbiol. 60, 51–66 (2006)

    CAS  Article  Google Scholar 

  24. Horoschak, K. D. & Moulder, J. W. Division of single host cells after infection with chlamydiae. Infect. Immun. 19, 281–286 (1978)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Oswald, E., Nougayrede, J. P., Taieb, F. & Sugai, M. Bacterial toxins that modulate host cell-cycle progression. Curr. Opin. Microbiol. 8, 83–91 (2005)

    CAS  Article  Google Scholar 

  26. Shafikhani, S. H. & Engel, J. Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps. Proc. Natl Acad. Sci. USA 103, 15605–15610 (2006)

    ADS  CAS  Article  Google Scholar 

  27. Lee, V. T., Anderson, D. M. & Schneewind, O. Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: One-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone. Mol. Microbiol. 28, 593–601 (1998)

    CAS  Article  Google Scholar 

  28. Day, J. B., Ferracci, F. & Plano, G. V. Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies. Mol. Microbiol. 47, 807–823 (2003)

    CAS  Article  Google Scholar 

  29. Chen, Y., Smith, M. R., Thirumalai, K. & Zychlinsky, A. A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J. 15, 3853–3860 (1996)

    CAS  Article  Google Scholar 

  30. Guichon, A., Hersh, D., Smith, M. R. & Zychlinsky, A. Structure-function analysis of the Shigella virulence factor IpaB. J. Bacteriol. 183, 1269–1276 (2001)

    CAS  Article  Google Scholar 

  31. Mumberg, D., Muller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995)

    CAS  Article  Google Scholar 

  32. Gietz, R. D. & Woods, R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002)

    CAS  Article  Google Scholar 

  33. Miller, R. K. Monitoring spindle assembly and disassembly in yeast by indirect immunofluorescence. Methods Mol. Biol. 241, 341–352 (2004)

    PubMed  Google Scholar 

  34. Day, A., Schneider, C. & Schneider, B. L. Yeast cell synchronization. Methods Mol. Biol. 241, 55–76 (2004)

    PubMed  Google Scholar 

  35. Zhang, H. & Siede, W. Analysis of the budding yeast Saccharomyces cerevisiae cell cycle by morphological criteria and flow cytometry. Methods Mol. Biol. 241, 77–91 (2004)

    PubMed  Google Scholar 

  36. Dorer, R. K. et al. A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes. Curr. Biol. 15, 1070–1076 (2005)

    CAS  Article  Google Scholar 

  37. Tugendreich, S. et al. A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays. Genome Res. 11, 1899–1912 (2001)

    CAS  Article  Google Scholar 

  38. Perkins, E. et al. Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast. Cancer Res. 61, 4175–4183 (2001)

    CAS  PubMed  Google Scholar 

Download references


We thank members of the Lesser and Lory laboratories for discussions, N. Slagowski of the Lesser laboratory for assistance in the yeast growth assay, C. Shamu and the members of the ICCB Screening facility at Harvard Medical School for granting access to chemical compounds and assistance with screening, and R. Dorer of the laboratory of A. Murray at Harvard University for sharing the drug sensitive yeast strain. We thank B. Kaltenboeck at Auburn University, V. Lee, Z. Balsara and M. N. Starnbach at Harvard Medical School for sharing BGMK cells, Y. entercolitica pYVe227 plasmid DNA, and C. trachomatis L2 genomic DNA.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Stephen Lory.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4 with Legends and Supplementary Tables 1-2 (PDF 635 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huang, J., Lesser, C. & Lory, S. The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth. Nature 456, 112–115 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing