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

Abstract

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.

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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.

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Acknowledgements

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.

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Correspondence to Stephen Lory.

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Huang, J., Lesser, C. & Lory, S. The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth. Nature 456, 112–115 (2008). https://doi.org/10.1038/nature07355

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