SCOT OUELLETTE, PhD
Role of the Clp Protease System in Chlamydial Growth and Pathogenesis
Chlamydia encodes two ClpP paralogs, which is somewhat unusual for a bacterium, and two different AAA+ ATPases (ClpX and ClpC). Given its developmental cycle that alternates between an EB and RB, we hypothesize that the Clp protease systems are critical in differentiation. To begin to study this, we bioinformatically analyzed each ClpP protein, monitored its transcript and protein expression during the development, and assessed effects of over-expression of each on chlamydial growth. We further assayed recombinant protein preps of each ClpP paralog for protease activity and observed that only ClpP2 was functional in vitro. ClpP2 was also activated by ACP1 derivatives, known to activate other ClpP homologs. We further screened a library of compounds based on the ACP1 scaffold to identify new candidates that blocked chlamydial growth. Most recently, we demonstrated unique functions for ClpX and ClpP2 in developmental cycle progression using a combination of over-expression and CRISPRi-mediated knockdown approaches. Current work is focused on substrate identification with an expectation that metabolic enzymes will be targeted for degradation as the RB converts to the metabolically inert EB.
Chlamydial transcriptional dynamics during productive and persistent growth
Chlamydiae have the ability to enter a non-dividing but viable growth state termed persistence in response to stress, particularly nutrient stress. Prior studies had suggested that Chlamydia engages a defined transcriptional response during persistence, but our data contradicted these findings since transcription was globally elevated, likely owing to the absence of a stringent response in this bacterium. We recently demonstrated that chlamydial transcription during interferon-g-mediated tryptophan limitation is linked to the tryptophan codon content of the gene and further showed a role for Rho-mediated transcriptional termination after ribosome stalling during tryptophan starvation. Most recently, we demonstrated the efficacy of bacterial tRNA synthetase inhibitors in inducing amino acid-specific starvation states to induce persistence. These tools should prove useful in combining genetic approaches with the study of persistence, which we demonstrated recently in collaboration with the Carabeo lab. Understanding how alterations in host cell and bacterial metabolism influences entry into the persistent state is a long-term goal of the lab.
- Bui, D.-C.^, Jorgenson, L.A.^, Ouellette, S.P., and Rucks, E.A. (2021). Eukaryotic SNARE VAMP3 dynamically interacts with multiple chlamydial inclusion membrane proteins. Immun. 89: e00409-20. (^Contributed equally.)
- Pokorzynski, N.D., Hatch, N.D., Ouellette, S.P., and Carabeo, R.A. (2021). The iron-dependent repressor YtgR is a tryptophan-dependent attenuator of the trpRBAoperon in Chlamydia trachomatis. Nature Comm. in press
- Wood, N.A., Blocker, A.M., Seleem, M.A., Conda-Sheridan, M., Fisher, D.J., and *Ouellette, S.P.(2020). The ClpX and ClpP2 orthologs of Chlamydia trachomatis perform discrete and essential functions in organism growth and development. mBio 11: e02016-20.
- Hatch, N.D., and *Ouellette, S.P.(2020). Inhibition of tRNA synthetases induces persistence in Chlamydia. Immun.88: e00943-19.
- Olson, M.G., *Ouellette, S.P., and *Rucks, E.A. (2020). A meta-analysis of affinity purification-mass spectrometry experimental systems used to identify eukaryotic and chlamydial proteins at the Chlamydia trachomatisinclusion membrane. Proteomics 212: 103595.
- Olson, M.G., Widner, R., Jorgenson, L.M., Lawrence, A., Lagundzin, D., Woods, N., *Ouellette, S.P., and *Rucks, E.A. (2019). Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatisInfected Human Cells. Immun. 87: e00537-19.
- *Ouellette, S.P., Messerli, P.J., Wood, N.A., and Hajovksy, H. (2018). Characterization of chlamydial Rho and the role of Rho-mediated transcriptional polarity during IFNg-mediated tryptophan limitation. Immun.86: e00240-18.
- *Ouellette, S.P., Rueden, K.J., and Rucks, E.A. (2016). Tryptophan codon-dependent transcription in Chlamydia pneumoniae during IFNg-mediated tryptophan limitation. Infect. Immun. 84: 2703-2713.