Abstract
Mycobacterium tuberculosis (Mtb) defends itself against host immunity and chemotherapy at several levels, including the repair or degradation of irreversibly oxidized proteins (IOPs). To investigate how Mtb deals with IOPs that can neither be repaired nor degraded, we used new chemical and biochemical probes and improved image analysis algorithms for time-lapse microscopy to reveal a defense against stationary phase stress, oxidants, and antibiotics--the sequestration of IOPs into aggregates in association with the chaperone ClpB, followed by the asymmetric distribution of aggregates within bacteria and between their progeny. Progeny born with minimal IOPs grew faster and better survived a subsequent antibiotic stress than their IOP-burdened sibs. ClpB-deficient Mtb had a marked recovery defect from stationary phase or antibiotic exposure and survived poorly in mice. Treatment of tuberculosis might be assisted by drugs that cripple the pathway by which Mtb buffers, sequesters, and asymmetrically distributes IOPs.
🔬 Techniques
🧬 Organisms
✨ Fluorophores
🧪 Sample Preparation
💻 Software Details
💾 Data Repositories
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📋 Methods
Procedures for oxidation of BSA, detection of carbonyls, cell fractionation, cross-linking, synthesis of probes for carbonyls, construction of strains, imaging, infection of mice and determination of MICs are described in the Extended Experimental Procedures.
Strains and culture conditions Table
S1 lists strains. Strains were cultured at 37°C in Middlebrook 7H9 with 0.2% glycerol, 0.5% bovine serum albumin fraction V (BSA), 0.02% tyloxapol, 0.2% dextrose, and 0.085% NaCl. Strains bearing antibiotic cassettes were cultured with hygromycin(50 μg/mL), zeocin (25 μg/mL), nourseothricin (25 μg/mL), or streptomycin (25 μg/mL).
Light microscopy and analysis
TLM was performed in a custom-built microfluidic device. Images were processed with the open-source programs ImageJ ( http://imagej.nih.gov/ij/ ) or Icy ( http://icy.bioimageanalysis.org/ ) using customized plugins, as detailed in Extended Experimental Procedures. Data were compiled using Matlab (The MathWorks).
Supplementary Material 1 2 3 4 5 6 7
📊 Figures
Figure 1
Proteomic analysis of Mtbu2019s IOP, see also Figure S1
(A) Haber-Weiss chemistry ((1) and (2)) drives OHu2022 generation and addition of carbonyls on amino acid side chains, e.g. an arginyl residue (3). (B) DNPH-reacted protein samples from Mtb in logarit...
Figure 2
ClpB largely co-localizes with IOP, see also Figures S2 and S3
(A) Distribution of ClpB-GFP (green) and carbonyls reactive with TR-ATPAH (red) in Mtb in logarithmic phase (LOG) and stationary phase (STAT). Scale bar, 2 microns for the last row, 4 microns in other...
Figure 3
ClpB-Dendra2 foci form in response to sublethal kanamycin
(A) Perfusion paradigm. 7H9, Middlebrook 7H9 medium. KAN, kanamycin (1 u03bcg/mL). (B) Representative image series for phases defined in (A). Numbers indicate time in hours. Scale bar, 2 u03bcm. Diffu...
Figure 4
Quantitative characterization of ClpBu2019s association with aggregates in response to kanamycin at the microcolony level, see also Figure S4
(A, C, D, F, G, H, I and J) Shaded areas indicate incubation of Msm with 1 u03bcg/mL kanamycin (panels A, C, D and F) or 0.1 u03bcg/mL (panels G to J). The same 154 microcolonies were analyzed in (A),...
Figure 5
Distribution of ClpB into aggregates within mycobacteria leads to asymmetric distribution of aggregates between their progeny
(A) Photomicrographic evidence. Aggregates accumulate at the cell pole in the parental Msm cell (M) in response to exposure to kanamycin. After removal of kanamycin, M divides. Descendant D1 inherits ...
Figure 6
Heterogeneous distribution of ClpB-GFP within mycobacteria and between their progeny in response to a sublethal concentration of INH
(A) Perfusion paradigm. 7H9, Middlebrook 7H9 medium. INH, isoniazid (40 u03bcg/mL). Shaded phases II and IV correspond to shading in panels (Cu2013E). (B) Representative image series for each phase in...
Figure 7
Contribution of ClpB to Mtbu2019s fitness, see also Figure S5
(A) Defective recovery of ClpB-deficient Mtb from stationary phase. Growth of H37Rv (circles), H37Rvu0394 clpB (squares) and the complemented strain H37Rvu0394 clpBu2237clpB (triangles) in 7H9 medium ...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
0 commentsNo comments yet. Be the first to start a discussion!
Leave a Comment