Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells - PubMed (original) (raw)

. 2015 Feb 11;17(2):178-90.

doi: 10.1016/j.chom.2014.12.008. Epub 2015 Jan 22.

Gang Lin 1, Neeraj Dhar 2, Nicolas Chenouard 3, Xiuju Jiang 1, Helene Botella 1, Tania Lupoli 1, Olivia Mariani 4, Guangli Yang 5, Ouathek Ouerfelli 5, Michael Unser 6, Dirk Schnappinger 1, John McKinney 2, Carl Nathan 7

Affiliations

Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells

Julien Vaubourgeix et al. Cell Host Microbe. 2015.

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.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Proteomic analysis of Mtb’s IOP, see also Figure S1

(A) Haber-Weiss chemistry ((1) and (2)) drives OH• generation and addition of carbonyls on amino acid side chains, e.g. an arginyl residue (3). (B) DNPH-reacted protein samples from Mtb in logarithmic phase (LP) from cultures that were: untreated or supplemented with NaNO2 (3 mM, pH 5.5), H2O2 (5 mM), or a combination of nitrite (0.5 mM) and H2O2 (2.5 mM) at pH 5.5; or subjected to starvation for 1 to 2 weeks in phosphate buffered saline (PBS) and H2O2 (5 mM); or from Mtb in stationary phase (SP) (left panel). Hydrazone derivatives were blotted with anti-DNP antibody. Ponceau red staining served as a loading control (right panel). MW, molecular weight controls that migrated in a single band on 5% SDS stacking gel. (C) Reaction of fluorophore-derivatized 15-amino-4,7,10,13-tetraoxapentadecanoic acid hydrazide (ATPAH) with carbonyls. R and R1 indicate the N- and C- termini of a protein. R2 represents FITC or Texas red. (D) Reaction of probe in (C) with oxidized BSA. The amount of staining (first 4 lanes of (D, left)) increased with the concentration of the oxidant pair FeCl3 and ascorbic acid, incubated for 12 h in the equimolar concentrations indicated in (D, right) with BSA. Staining also increased with the quantity of oxidized BSA (125, 250 and 500 ng) as indicated by the open, gray and black circles below the 3 lanes of (D, bottom left) and quantified in (D, right). (E) Intracellular distribution of HupB, a carbonylated protein, as a fusion with Dendra2 in relation to its carbonylation status (TR-APTAH stain) and the nucleoid (Hoechst) in logarithmic (LOG) and stationary phase (STAT). Intensity of fluorescence (AU) in the 3 channels is quantified along the longitudinal axis (yellow) of representative cells. Inspection of z-planes showed that bright TR-APTAH staining of some LOG cells came from the cell surface. Scale bar, 2 microns.

Figure 2

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 micrographs. (B) Protein carbonylation in Msm subjected to indicated concentrations of kanamycin (μg/mL) prior to fractionation into soluble (SOL) and sedimentable (SED) fractions and reacted with FITC-APTAH prior to SDS-PAGE. Gels were stained with Coomassie (two gels on left) or laser-illuminated (480 nm) (two gels on right). Higher Mr species marked “IOP” may correspond to cross-linked aggregates. Number in white indicate the intensity of the FITC-APTAH signal (AU). (C) Redistribution of ClpB-GFP into the sedimentable fraction following exposure of Msm to kanamycin. ΔClpBsm∷ClpBsm-GFP cells were exposed for 3 h to kanamycin at indicated concentrations prior to fractionation and immunoblotting. (D) Association of ClpB with higher-Mr protein complexes following kanamycin treatment of Msm. ΔClpBsm∷ClpBsm cells were treated with 1 μg/mL of kanamycin for 30 or 90 min and then exposed or not to the cross-linking agent DSG. ClpB-GFP was detected by immunoblot in unfractionated extracts. The arrow indicates the slowest bands emerging preferentially upon kanamycin treatment. (E) Redistribution of diffuse ClpB-GFP to aggregates in response to sublethal kanamycin. Scale bar, 4 μm. (F) Entry of ClpB into focal structures in kanamycin-treated Msm. A Msm∷ClpB-Dendra2 cell that was treated with 1 μg/mL kanamycin for 2 h contained two focal accumulations of ClpB (open circle and open square, top panel). A region distant from both foci (cross) was illuminated with a 405-nm laser. Dendra2 fluoresced in the green channel (“Green”) before photoconversion (BPC), and in the red channel (“Red”) after photoconversion (APC). Micrographs in the red channel were taken at time 0 and 5, 9, 13, 17 and 21 sec after photoconversion (F, right). (G) Quantitative representation of the entry of ClpB from the irradiated site in the cell in (F) into the foci indicated by the square and the circle in (F). New molecules of ClpB joined the nearer focus first.

Figure 3

Figure 3. ClpB-Dendra2 foci form in response to sublethal kanamycin

(A) Perfusion paradigm. 7H9, Middlebrook 7H9 medium. KAN, kanamycin (1 μg/mL). (B) Representative image series for phases defined in (A). Numbers indicate time in hours. Scale bar, 2 μm. Diffuse ClpB redistributed into aggregates upon exposure of Msm to kanamycin. Aggregates later decreased in number. 256 micrographs were recorded over 64 h at constant Illumination.

Figure 4

Figure 4. Quantitative characterization of ClpB’s 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 μg/mL kanamycin (panels A, C, D and F) or 0.1 μg/mL (panels G to J). The same 154 microcolonies were analyzed in (A), (C), (D), (E) and (F). Results in (A), (C), (D), (E) and (F) are compiled from 3 experiments. They are representative of results in >15 additional experiments conducted with variations in the timing of perfusion phases, doses of kanamycin or choice of fluorescent tag. (A) Numbers of aggregates per bacterial pixel over time (blue circles) and numbers of aggregates per microcolony (red circles). (B) Representative image series (Δt = 30 min) of 2 Δ_clpBSm_∷_clpB_-dendra2 cells over 4 h following exposure to 1 μg/mL of kanamycin. Quantitated, false-color fluorescence images superimposed on phase contrast images illustrate an initially relatively diffuse distribution of ClpB-GFP, followed by its accumulation into aggregates in conjunction with its disappearance from the intervening volume of the cell. The upper cell develops 2 aggregates; the lower cell develops 3 that fuse into 1. (C) Mean fluorescence intensity of microcolonies. The mean intensity of fluorescence of bacterial pixels (navy blue) was corrected for photobleaching as in Figure S5A. (D) Numbers of aggregates of different sizes (S) over time. (E) Tendency of ClpB-associated aggregates to collect at a pole. The histogram represents the frequency distribution (y-axis) of aggregates’ localization in Δ_clpBSm_∷_clpB_-dendra2 as a function of time in relation to kanamycin exposure on the long axis of each cell, where distances (D) of 0.50 and 1.00 on the x-axis represent mid-cell and a cell pole, respectively. T = time period analyzed in hours. (F) Growth of microcolonies. The number of bacterial pixels (red circles) was summed over time. (G to J) Numbers of aggregates per bacterial pixel over time (purple circles). Expression of fluorescent controls was induced by pulse perfusion with Atc (dashed red line; 200 ng/mL). (G) Failure of ClpBY251A-GFP to enter aggregates in response to 0.1 μg/mL of kanamycin. Transient appearance of fluorescent patches after Atc was attributable to the fluorescence of Atc. (H) Tendency of ClpB-GFP expressed in Msm with strictly similar genetic background as in (G) to enter aggregates in response to 0.1 μg/mL of kanamycin. (I) Failure of PrcA-mCherry to enter aggregates in response to 0.1 μg/mL of kanamycin. Msm expressed PrcA-mCherry and PrcB (heptamers in stacks of 4) in response to pulse perfusion with Atc. (J) Failure of GFP not fused with ClpB to enter aggregates in response to 0.1 μg/mL of kanamycin.

Figure 5

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 no aggregates and survives; descendant D2 inherits the ClpB-GFP aggregate and lyses (L). Numbers indicate time in hours. Scale bar, 2 μm. (B–E). Fluctuation in number of aggregates in individual Msm cells and relation of aggregate burden to growth rate. Top panels, aggregate burden (sum of Sa), representing total fluorescent intensity (AU) of aggregates. Lower panels, number of aggregates (dashed lines) and cell length (μm). Black line, parental cell; red and blue lines, progeny. (B) Example of a cell that failed to give IOP to progeny and died, losing fluorescence. (C, D) Examples of cells whose progeny received markedly different burdens of IOP, with the more heavily burdened descendant dying ((C); red dot denotes lysis at the time of division of the parental cell) or growing much more slowly than its less burdened sibling (D). (E) Example of cells that bestowed nearly equal burdens of IOP on their progeny, whose growth rates were then almost the same. (F) Relative mean elongation velocity of progeny of stressed parents correlated negatively with their cumulative Sa and with their length at birth (see text). (G) Ability of cells to divide after a second exposure to kanamycin correlated with their rate of elongation (Y-axis) before the second exposure. X-axis: “0” denotes cells that failed to divide following the second kanamycin exposure; “1” indicates cells that divided at least once during the subsequent drug-free perfusion.

Figure 6

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 μg/mL). Shaded phases II and IV correspond to shading in panels (C–E). (B) Representative image series for each phase in (A). Numbers indicate time in hours. Scale bar, 4 μm. False-colored fluorescence intensity, corrected for photobleaching, Decimals denote lineages of parental cells (M) and their descendants (D). Thus, M1’s descendant D1.1 gave rise to D1.1.0, which inherited few patches of ClpB, and D1.1.1, which inherited most of the patches of ClpB. Likewise, D2.0 inherited almost no detectable ClpB from M2, while D2.1 (see inset) inherited almost all of it. Results were similar for the descendants of M3. (C) Relation of microcolony growth to expression of ClpB-GFP. Median growth of 79 microcolonies (yellow circles; total bacterial pixels) is shown on a linear scale in relation to incubation with INH. Summed intensity of ClpB-GFP fluorescence is denoted on a linear scale by blue circles (AU). Total expression of ClpB-GFP increased much more slowly than microcolony biomass, but increased fastest when colony growth slowed. (D) Quantitative characterization of formation of ClpB-GFP into patches following exposure of Msm microcolonies to INH. As mean fluorescence (fluorescence intensity divided by bacterial pixels; blue circles) of the microcolonies decreased, the median coefficient of variation (σ/μ) of the mean fluorescence increased (red circles). Significance was evaluated by Kolmogorov-Smirnov test for continuous distribution as the null hypothesis. (E) Quantitative characterization of asymmetric distribution of patched ClpB to progeny following exposure of Msm to sublethal INH. Each dot corresponds to the absolute value of the difference in ClpB fluorescence mean intensities at birth (|ΔMD1-|D2)|) between members of a progeny pair, ID1 and ID2. This is compared to the mean (red line) of the difference in background fluorescence, calculated as the difference in fluorescence mean intensities of two adjacent areas that were external to cells, identical to each other in size, and similar in size to a bacterium. This background value was calculated every 15 min over 23.5 h for 17 movies from 3 independent experiments. Blue line indicates a confidence level > 99.9% (p < 0.004).

Figure 7

Figure 7. Contribution of ClpB to Mtb’s fitness, see also Figure S5

(A) Defective recovery of ClpB-deficient Mtb from stationary phase. Growth of H37Rv (circles), H37RvΔ_clpB_ (squares) and the complemented strain H37RvΔ_clpB∷clpB_ (triangles) in 7H9 medium using inocula from logarithmic phase (black), early stationary phase (gray) or late stationary phase (open symbols). Mean ± SD of 2 experiments, each in duplicate. (B) Excessive accumulation of carbonyls in ClpB-deficient Mtb in stationary phase. ATPAH reactivity (arbitrary fluorescence units) was compared in late stationary phase in H37RvΔ_clpB_ and H37RvΔ_clpB∷clpB_ cells relative to that in wild type H37Rv, which was set to 1. Mean ± SD of 4 experiments, each in duplicate. (C) Attenuation of ClpB-deficient Mtb in mice. Colony-forming units (CFU) of H37Rv (circles), H37RvΔ_clpB_ (squares) and H37RvΔ_clpB∷clpB_ (triangles) in lungs of C75BL/6 mice at the indicated times after aerosol infection. Means ± SD for 5 mice per time point from one experiment representative of 2. (D) Diminished histopathology caused by ClpB-deficient Mtb. Sections of lungs collected at day 120 from mice infected with the indicated strains were stained with hematoxylin and eosin. Photographs show the sections at their actual size.

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