STRUCTURAL AND FUNCTIONAL STUDIES OF THREE PROTEINS OF UNKNOWN FUNCTION ENCODED BY CHLAMYDIA TRACHOMATIS

Infections by chlamydial species are of significant impact to global public health, causing sexually transmitted infections, blinding trachoma and pneumonia. Despite its importance, there are many aspects of chlamydial biology that are not completely understood, including the mechanisms by which it infects, persists and replicates in its host cells. The reason for this ignorance of basic chlamydial biological processes is because there is an abundance of Open Reading Frames (ORFs) of unknown function present in chlamydial genomes, almost 30% of the entire genome in many species. This is likely due to the relatively large phylogenetic distance between Chlamydiae and better-understood bacteria such as E. coli and B. subtilis. Current strategies of genome annotation rely on the presence of homology to genes of known function and these approaches have not been effective in annotating chlamydial genomes. In an effort gain insight into the function of these chlamydial ORFs of unknown function, I utilized structural information (both computational and experimentally derived) about three proteins of interest. Based on these structural studies, hypotheses concerning the functions of these proteins were formed and then tested. Together, my findings not only provide valuable information about these proteins of unknown function, but they also serve to demonstrate both the strengths and shortcomings of the overall approach of utilizing structural information for functional prediction. One example of this approach is my work on the chlamydial ORF CT296. Although this protein was annotated as having an unknown function (due to insignificant homology to proteins of known function), it had been experimentally characterized as an iron-dependent transcription factor. Having an experimentally characterized function allowed me to test my approach of utilizing structural information to predict function on a protein with a relatively well-understood function. Surprisingly, structural information of this protein suggested that it functions as a Fe(II) 2-oxoglutarate-dependent enzyme and not as a transcription factor. Subsequent functional analyses of the protein were unable to reproduce previous reports of its DNA binding. Together, my findings suggest that this protein may not function as a transcription factor. A second example of my structure-function approach was applied to the chlamydial protein CT584. This protein was first experimentally described as interacting with the chlamydial Type Three Secretion System (T3SS) needle protein in an interactome study. This observation, combined with a subsequent biophysical characterization of the protein lead to an initial hypothesis that the protein may be a chlamydial T3SS needle tip protein. However, results of structural studies on the protein reveal a structure that is not similar to any of the known T3SS needle tip proteins. Additionally, functional studies on the protein focusing on identifying its localization on chlamydial organisms revealed localization patterns not consistent with its proposed role as a T3SS needle protein. Together, my studies suggest that this protein may not function as a needle tip protein. A final example of the utility of structural information for informing function concerns chlamydial ORF CT009. This protein was annotated in many chlamydial species as a protein of unknown function; however, bioinformatics analyses had identified it as a helix-turn-helix containing transcription factor. Results of computational and experimental structures of this protein show structural similarity to the protein RodZ, a key component of the bacterial morphogenic complex. Subsequent functional analyses of CT009 demonstrate that this protein has the characteristics of a chlamydial homolog to RodZ

Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

This is the published version. Copyright American Chemical SocietyChlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Likemany Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche, and possibly cellular exit. Computational structure prediction indicated that ORF CT584 is homologous to other T3SS needle tip proteins. Tip proteins have been shown to be localized to the extracellular end of the T3SS needle and play a key role in controlling secretion of effector proteins. We have previously demonstrated that T3SS needle tip proteins from different bacteria share many biophysical characteristics. To support the hypothesis that CT584 is a T3SS needle tip protein, biophysical properties of CT584 were explored as a function of pH and temperature, using spectroscopic techniques. Far-UV circular dichroism, Fourier transform infrared spectroscopy, UV absorbance spectroscopy, ANS extrinsic fluorescence, turbidity, right angle static light scattering, and analytical ultracentrifugation were all employed to monitor the secondary, tertiary, quaternary, and aggregation behavior of this protein. An empirical phase diagram approach is also employed to facilitate such comparisons. These analyses demonstrate that CT584 shares many biophysical characteristics with other T3SS needle tip proteins. These data support the hypothesis that CT584 is a member of the same functional family, although future biologic analyses are required

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

This is the peer reviewed version of the following article: Kemege, K. E., Hickey, J. M., Barta, M. L., Wickstrum, J., Balwalli, N., Lovell, S., Battaile, K. P. and Hefty, P. S. (2015), Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB. Molecular Microbiology, 95: 365–382. doi:10.1111/mmi.12855, which has been published in final form at http://doi.org/10.1111/mmi.12855. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis, and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient E. coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia

Structure of CT584 from Chlamydia trachomatis refined to 3.05 Å resolution

Chlamydia trachomatis is a major cause of various diseases, including blinding trachoma and pelvic inflammatory disease, and is the leading reported sexually transmitted bacterial infection worldwide. All pathogenic Chlamydiae spp. utilize a supramolecular syringe, or type III secretion system (T3SS), to inject proteins into their obligate host in order to propagate infection. Here, the structure of CT584, a T3SS-associated protein, that has been refined to a resolution of 3.05 Å is reported. The CT584 structure is a hexamer comprised of a trimer of dimers. The structure shares a high degree of similarity to the recently reported structure of an orthologous protein, Cpn0803, from Chlamydia pneumoniae, which highlights the highly conserved nature of this protein across these chlamydial species, despite different tissue tropism and disease pathology