Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology

An Examination of Not-For-Profit Stakeholder Networks for Relationship Management: A Small-Scale Analysis on Social Media

Using a small-scale descriptive network analysis approach, this study highlights the importance of stakeholder networks for identifying valuable stakeholders and the management of existing stakeholders in the context of mental health not-for-profit services. We extract network data from the social media brand pages of three health service organizations from the U.S., U.K., and Australia, to visually map networks of 579 social media brand pages (represented by nodes), connected by 5,600 edges. This network data is analyzed using a collection of popular graph analysis techniques to assess the differences in the way each of the service organizations manage stakeholder networks. We also compare node meta-information against basic topology measures to emphasize the importance of effectively managing relationships with stakeholders who have large external audiences. Implications and future research directions are also discussed

Proteomic profiling of P750-clpP1P2DAS in the presence and absence of ATc reveals a wide array of potential Clp protease substrates.

<p>(A) In triplicate, P750-clpP1P2DAS was grown for 48 hours in the absence, denoted “wt”, or presence of ATc (1.5 µg/mL), denoted “mut”, from a starting OD₆₀₀ of 0.02. Immunoblotting of protein lysates with α-ClpP2 and α-RpoB (loading control) demonstrates degree of ClpP2-DAS depletion in mut cells. Samples were then used for TMT₆ MS3-based quantitative proteomics. The specific TMT reagent used for each condition is listed under the immunoblot. (B) Normalized, summed intensities for all quantified proteins was used to perform Pearson correlational hierarchical clustering of biological replicates. (C) The Log2 ratio of average mutant protein intensity to average wildtype protein intensity plotted against the p-value determined by t-test, grouping the three biological replicates. The threshold for over-representation was set at an average ratio of greater than equal to 2, while the cut-off for under-representation was 0.5. In both instances, p-values below 0.01 were deemed significant. Proteins considered for further analysis are denoted in red. (D) The relative quantity of specific proteins plotted across the six TMT channels, for highly (left) and moderately (center) over-represented, and under-represented (right) proteins in the mutant versus wildtype conditions.</p

Validation of proteomic hits reveals that WhiB1 and CarD are likely Clp protease substrates.

<p>(A) Quantitative PCR to determine transcript levels of over-represented proteins upon depletion of Clp protease using RNA generated from Mtb P750-clpP1P2DAS after growth for 48 h in the presence or absence of ATc (1.5 µg/mL). Relative standard curves were generated for each probe set, and sigA transcript was used as an endogenous control. Data are represented as mean fold change, normalized to transcript in (−) ATc cultures +/− SEM of technical replicates. Protein ratios are derived from TMT experiments described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003994#ppat-1003994-g002" target="_blank">Figure 2</a>, and represented as average ratios +/− standard deviation of biological replicates (B) Fluorescence (485/538) was measured for N- and C-terminal GFP fusions constructed for WhiB1 (left) and CarD (right), and induced for 8 hours in wildtype or clpP2-ID Msm with ATc (100 ng/mL). In clpP2-ID, ATc simultaneously induced fusion protein production and degradation of ClpP2. (C) Fluorescence (485/538) was measured for GFP fusions bearing a variable number of C-terminal residues from WhiB1 in clpP2-ID Msm, grown in the absence or presence of ATc (100 ng/mL) for 8 hours. In (B) and (C), data are represented as mean RFU +/− standard deviation of biological replicates. Asterisks denote a p-value <0.05 determined by t-test.</p

Blocking Clp-dependent degradation of WhiB1 is toxic in mycobacteria.

<p>(A) Inducible production of GFP-WhiB1, WhiB1-GFP, and WhiB1wt in Msm demonstrates growth inhibition of WhiB1-GFP producing bacteria. Over-production was induced with ATc (100 ng/mL). (B) Growth curves of strains producing WhiB1 constructs in the presence of ATc (100 ng/mL). As a control, strains producing WhiB1wt were grown in the absence of inducer. Data are represented as mean OD₆₀₀ +/− standard deviation of biological replicates. (C) Quantitative PCR, using probe sets that hybridize to the whiB1 5′utr, to determine transcriptional repression at the endogenous <i>whiB1</i> locus in wildtype Msm, or Msm inducibly expressing <i>gfp-whiB1</i> or <i>whiB1-gfp</i>. RNA was isolated from cultures grown for 6 hours from a starting OD₆₀₀ of 0.06 in the presence of the inducer ATc (100 ng/mL). Relative standard curves were generated for each probe set, and sigA transcript was used as an endogenous control. Data are represented as mean fold change, normalized to transcript in wildtype cultures +/− SEM of technical replicates. (D) Promoter reporters were constructed fusing the 500 bp upstream of whiB1 to luciferase. Luminescence (RLU, 100 ms exposure, grey bars) was measured in wildtype Msm, or Msm inducibly producing GFP-WhiB1 or WhiB1-GFP. Amounts of the fusion proteins were monitored by fluorescence (RFU, 485/538, black bars). Data are represented as RFU or RLU +/− standard deviation of biological replicates. (E) GFP whiB1 fusions and wildtype whiB1 were cloned into integrative plasmids, in which expression was driven by the native <i>whiB1</i> promoter. Constructs were transformed into Msm, and transformation efficiency was determined by calculating colony forming units (CFU) obtained per ng DNA. In (C), (D), and (E), asterisks denote a p-value <0.05, determined by t-test.</p