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

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dualcontrol genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies

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

Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection

Despite the worldwide efforts to avoid disease progression of COVID-19 into a severe acute respiratory syndrome and avoid its severe impact on health systems; the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are required to meet the worldwide demand: recombinant antibodies such as alpaca Nanobodies fulfill these requirements. Here, we develop a fast track for nanobody isolation against the receptor-binding-domain (RBD) SARS-CoV-2 Spike protein following an optimized immunization, efficient construction of the VHH library for E. coli surface display, and single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. Following this procedure, we isolate and characterize an alpaca Nanobody against Spike RBD of SARS-CoV-2 in the sub-nanomolar range.N