Goal 1. Identify and validate rapidly degraded proteins in wildtype Brucella abortus.
My lab has developed an approach using quantitative mass spectrometry and translation inhibitors to characterize protein degradation in bacteria. Collaborating with the Roop lab at East Carolina University, we have used this platform to generate a first-pass snapshot of proteome dynamics in Brucella abortus 2308. The Roop lab performed the translational shutoff experiment, then sent us inactivated, denatured cell pellets with 3 replicates of 0 minutes and 90 minutes post-shutoff. We then extracted proteins from these samples, trypsinized, fractionated, labeled with isobaric tags and performed TMT-based quantitative mass spectrometry. Using ThermoFisher Proteome Discoverer, we identified 1280 proteins with high confidence, then evaluated the loss of proteins over the shutoff using multiple testing corrected statistical methods suitable for this analysis.
This preliminary data set shows immense promise. It identifies many potential substrates that satisfy our criteria of statistical significance and fold-change. Of these potential substrates, we see CtrA and CpdR, known substrates of the ClpXP in Caulobacter (Jenal and Fuchs, 1999; Iniesta, et al. 2008), and SciP, an established substrate of the Lon protease in Caulobacter (Gora, et al. 2013). We also identify Lon as a potential degraded substrate, consistent with our recent findings of Lon instability in Caulobacter (Barros, et al. 2020). As examples of other potential substrates, in Figure 2 we also show BAB_RS27170 (also known as BAB2_0170), a small protein shown to be important for Brucella fitness (Sun, et al. 2012) and PicC (BAB_RS23615), an essential gene of unknown function found next to CtrA (Sternon, et al. 2018). We also identify stable proteins such as ClpX and RpsT, neither of which have been shown to be degraded in any bacterial systems.
Our next steps for Goal 1 are to validate the degradation of these substrates by i) purifying candidate proteins and testing their degradation in vitro, and ii) using westerns to test their turnover in vivo. The outcomes of this goal, combined with the ones laid out in goal 2 below will synergize to create a validated dataset that will be searchable and open access as described in Goal 3.
Goal 2. Determine the role of proteases, adaptors and conditions in proteome dynamics.
Based on the same approach described in Goal 1, we will use Brucella variants that lack either adaptors (CpdR and RcdA) or proteases (Lon) and chart proteome dynamics in these strains. Our hypothesis is that substrates which are rapidly turned over in wildtypestrains and stabilized in one of these variants are likely targets of those components in vivo. For example, degradation of CtrA occurs through the ClpXP complex and relies on the CpdR adaptor as shown by Willet, et al. We would predict that a proteome dynamics survey in ΔcpdR Brucella would show stabilization of CtrA compared to what we see in the wildtype data (Figure 2). Identifying other substrates stabilized similarly will allow us to determine other CpdR-dependent substrates or targets of the Lon protease. Because ClpXP is essential in Brucella (Roop lab, unpublished; Sternon, et al. 2018) we will use a dominant negative ClpX mutant that has been shown to inhibit native ClpX activity when expressed in trans (Jenal and Fuchs, 1998).
We will also use different stress conditions such as high heat, starvation and oxidative damage to determine how proteome dynamics change in these different conditions. Similar to how we plan to validate substrates from Goal 1, we will identify interesting candidates from this goal and validate as well.
Goal 3. Develop a searchable open-access database resource.
Because this data will be of value to all those studying Brucella, the last goal of this proposal is to develop a public searchable database that can be explored by users. We have already created a Shiny application on our departmental server to make similar data that we have collected in Caulobacter searchable by our lab and collaborators (Figure 3). Based off our success as shown by this example in Figure 3, our plan is to incorporate the proteome dynamics data that we will produce in Goals 1 and 2 along with existing published data on gene fitness, essentiality and expression that will serve as a resource for the Brucella community.
