Category B. Infectious Diseases and Vaccine Discovery
B1. Hypothetical Protein CT398 (CdsZ) from Chlamydia trachomatis Interacts with σ54 (RpoN) and the Type III Secretion Export System
Michael L. Barta1, Frances S. Mandelbaum1, Jason R. Wickstrum1, Scott Lovell2 , Kevin P. Battaile3, P. Scott Hefty1
1Department of Molecular Biosciences, University of Kansas
2Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas
3IMCA-CAT, Hauptman-Woodward Medical Research Institute
The relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (hypothetical proteins) is a broad challenge for bacteriology. The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function within the medically important, obligate intracellular human pathogen Chlamydia trachomatis. A crystal structure of CT398 was determined that displayed a high degree of structural similarity to FlgZ (Flagellar-associated zinc-ribbon domain protein) from Helicobacter pylori. This observation suggested putative protein partners for CT398 within Chlamydia, which were then experimentally confirmed. These partners included the alternative sigma factor RpoN (σ54) and two paralogous Type III Secretion System (T3SS) ATPase regulators (CdsL and FliH), among others. Additional interactions between flagellar orthologs and NF-T3SS export proteins were identified in order to gain insights into why flagellar genes would be retained in a non-motile intracellular organism. Finally, transformation of a pair of inducible expression vectors (affinity-tagged CT398 and RpoN) into Chlamydia was performed. Overexpression of CT398 revealed that growth of Chlamydia was altered and type III secretion was potentially disrupted. Overexpression and co-immunoprecipitation of RpoN demonstrated that CT398 associates with RNA polymerase holoenzyme (σ54 + core). Based upon these studies we propose that CT398 be named CdsZ (Contact dependent secretion zinc-ribbon domain protein). All together, this study suggests that CT398 functions as a protein partner in several key areas of chlamydial biology and pathogenesis, including as a chaperone of RpoN, modulating transcription in a currently unknown manner, and potentially in targeting mRNAs to the T3SS export apparatus for coupled translation and secretion.
B2. Identifying small molecule inhibitor binding site of AraC family regulators
Jiaqin Li, Graham Wehmeyer, Jeff M. Skredenske, Scott Lovell, Kevin P. Battaile, Susan M. Egan
Department of Molecular Biosciences, University of Kansas, Lawrence, KS
Protein members of the AraC family of bacterial transcriptional activators are required for expression of virulence factors by many bacterial pathogens, and thereby have great potential as targets for development of novel antibacterial agents. A small molecule inhibitor, SE-1, has been previously identified as an effective inhibitor of the AraC family protein RhaS. SE-1 blocks DNA binding and transcription activation by RhaS. SE-1 also blocks transcription activation by the DNA-binding domain of RhaS, indicating that this domain is the target of SE-1 inhibition. Site-directed mutagenesis of the RhaS DNA-binding domain was performed to test the binding site predicted by a docking model of SE-1 with RhaS-DBD. The results showed that the variant RhaS S249R was less sensitive than wild-type RhaS to inhibition by SE-1 in an in vivo reporter assay. This suggests that RhaS residue S249 is at or near the binding site for SE-1 on the RhaS DNA-binding domain. Structural studies of SE-1 binding to the RhaS DNA-binding domain have not been successful to date, so we have turned our attention to the ToxT protein. ToxT is an AraC family transcriptional activator that controls the expression of key virulence factors in Vibrio cholerae, the causative agent of Cholera. A crystal structure is available for ToxT, suggesting it will likely be more amenable for structural studies than RhaS. In vitro DNA binding assays with purified ToxT demonstrated that SE-1 inhibited DNA binding by ToxT. We have not yet obtained a structure of ToxT in a complex with SE-1, however, we have obtained crystals of ToxT, and have solved the structure to 1.65Å resolution. We will continue to optimize crystallization conditions to obtain a structure of the ToxT-SE-1 complex. In the meantime, our ToxT structure reveals details of the protein structure that were not well resolved in the previous structure, including a ten residue loop that was not previously modeled.
B3. Overexpression of the alternate sigma factor σ28 in Chlamydia trachomatis
Jason Wickstrum, Frances Mandelbaum, and P. Scott Hefty
Department of Molecular Biosciences, University of Kansas, Lawrence, KS.
Chlamydia trachomatis has only three known sigma factors for transcription initiation: σ66 (a σ70 homolog) and the alternate sigma factors σ54 and σ28. Potential target promoters for σ54 and σ28 have been predicted by sequence analysis, but the functions of the two alternate sigma factors in Chlamydia are not well understood. In this study, expression of σ28 was induced from a shuttle vector in vivo and the resulting change in the transcriptional profile of C. trachomatis was analyzed by RNA sequencing and quantitative PCR. Transcription of two genes, hctB (encodes a histone-like protein) and tsp (encodes a protease of unknown function), increased more than 30-fold. Microscopy analysis after induction showed evidence of potential premature nucleoid condensation, which is likely due to the increased expression of hctB. Other genes were upregulated after induction of σ28, but to a much lesser extent (2 – 3 fold), and most of those genes encode proteins of unknown function.