Fig 1.
Three-dimensional structure of Rv1466 (SufT).
Mtb SufT like other DUF59 proteins contains a small surface of conserved homology. Ribbon diagrams are shown for the Bacillus anthracis YitW (PDB ID: 3LNO); Mtb SufT (PDB ID: 5IRD); Thermotoga maritima TM0487 (PDB ID: 1UWD); and Homo sapiens FAM96a (PDB ID: 2M5H). The side chains of the conserved D, E, T, and C (red encircled) residues are highlighted. Oxygen, sulfur, and nitrogen atoms are represented with red, yellow, and blue color, respectively. Distance in Angstrom (Å) is represented between the side-chain hydroxyl of the T, D, and E residues. A proline (P25 in Mtb) present in the motif as DPE (blue encircled) and the consecutive proline (P63 in Mtb) next to the C residue are represented in the Mtb SufT structure.
Fig 2.
SufT interacts with SufS and SufU of SUF system.
(A) Model illustrating the principle of M-PFC. Two independent plasmid constructs for candidate proteins X and Y are shown in fusion to complementary mDHFR fragments F [1, 2] and F [3], respectively. Co-transformation of XF [1, 2] and YF [3] fusions in Msm results in the functional reconstitution of mDHFR activity and subsequent growth on TRIM (0 and 25 μg/mL; concentration) plates, whereas proteins that do not interact will not reconstitute F [1, 2] and F [3] and consequently no growth on TRIM plates. Components of vectors, aph confers resistance to KAN; hyg confers resistance to HYG; hsp60p is the hsp60 promoter; oriM is the origin of replication for propagation in mycobacteria; oriE is the origin of replication for propagation in E. coli; and int and attP are the integrase and phage attachment sites, respectively, from mycobacteriophage L5. (B) M-PFC based demonstration of C62 dependent specific protein-protein interaction of SufT with SufT, SufU and SufS. Msm as a host was co-transformed with plasmid construct expressing 1) SufT[F3]/GCN4[F1,2] (negative control), 2) GCN4[F1,2]/GCN4[F3] (positive control), 3) SufTC62A[F3]/SufT[F1,2], 4) SufT[F3]/SufT[F1,2], 5) SufTC62A[F3]/SufU[F1,2], 6) SufT[F3]/SufU[F1,2], 7) SufTC62A[F3]/SufS[F1,2] and 8) SufT[F3]/SufS[F1,2]. M-PFC experiments were repeated thrice, one representative image is shown here.
Fig 3.
SufT interacts with 4Fe-4S cluster containing proteins- SufR and Aconitase.
(A) M-PFC based demonstration of specific protein-protein interaction of SufT with Acn and SufR (4Fe-4S proteins), but not with SufTC62A. Construct 1) SufT[F3]/Acn[F1,2], 2) SufTC62A[F3]/Acn[F1,2], 3) SufT[F3]/SufR[F1,2], 4) SufTC62A[F3]/SufR[F1,2], 5) CFT10[F1,2]/ESAT6[F3] (positive control), and 6) empty vectors expressing [F3] and [F1,2] only (negative controls) were co-transformed in Msm and growth was scored on TRIM containing 7H11 plates. (B) The Mtb strain expressing pRH2502 (ATc inducible dCas9- vector control) and SufT specific gRNA in pRH2521 vectors (SufT-KD) was exposed to ATc (200 ng/mL) for 24 h and 48 h and the expression of sufT was monitored by RT-qPCR. (C) The SufT-KD strain was treated with 200 ng/mL of ATc and intracellular levels of SufT was determined at the indicated time points by immunoblotting using anti-SufT. Image density was analyzed with ImageJ software. Aconitase (Acn; 102 KDa) is used as loading control. (D) (top panel) Western blot of SufT and Acn from SufT-KD and vector control (with (+) and without (-) ATc) lysate. (lower panel) Immuno-precipitation of Acn from SufT KD (+ and − ATc) lysate with Anti-SufT IgG as well as rabbit Isotype IgG (Iso) as negative control. All M-PFC and western blot experiments were performed in triplicate, one representative image is shown here.
Fig 4.
SufT participates in Fe-S cluster biogenesis and repair.
(A) Western blot quantification of reduced SufT protein level (green bars) and decreased aconitase (Acn) activity (brown bars) was shown in SufT-KD strain, with increasing concentration (ng/mL) of ATc (0, 50, 100, 200 and 400). Level of SufT and enzymatic activity of Acn are normalized to Acn abundance from western blot. B) RT-qPCR based expression level of the suf operon (sufR, sufB, sufD, sufC, sufS, sufU), iscS, and anti-oxidant machinery (katG, sodA and ahpC) in SufT-KD strain. Horizontal dotted line indicates expression level below 1.5-fold considered as basal level. Data are shown as mean ±SD from triplicate reaction. (C) Acn activity was measured from cell free lysates derived from control and SufT-KD strains exposed to ATc (200 ng/mL) for 72 h and subjected to Fe-S reconstituted (Fe2+) and Dipyridyl (DIP) treatment. UT; untreated. D) Acn activity of UT, H2O2 and H2O2+Catalase treated cell lysates from control and SufT-KD strains were measured after 48 h of ATc (200 ng/mL) treatment. Student’s t test (two tailed) was performed here with p values, *p = 0.05,**p = 0.01 and ***p = 0.001.
Fig 5.
Depletion of SufT results in deregulation of metabolites dependent on Fe-S cluster proteins.
Single column based LC MS/MS analysis of (A) OBHA derivatized TCA metabolites, underivatized measurement of purines, SAM, Cystathione (CTH), SAH and (B) Amino acids of SufT-KD strain post 72 h of ATc (200 ng/mL) treatment. Experiments were performed in biological triplicate and p value was calculated by two-way ANOVA multiple comparison test. ns p ≥ 0.05, *p ≤0.05, **p ≤0.01, ***p ≤0.001.
Fig 6.
Depletion of SufT results in reduced OCR and ECAR.
(A) A change in % oxygen consumption rate (% OCR) and (B) % extra cellular acidification rate (% ECAR) of SufT-KD strain precultured for 72 h in the presence (+ATc) or absence (–ATc) of ATc and was measured after injection of 5 mM glucose (Glc) and 5 μM CCCP indicated by dotted lines. The uncoupler CCCP was used to determine the spare respiratory capacity (SRC). (C) Decrease in 20% SRC of SufT-KD+ATc with respect to SufT-KD-ATc. Percentage SRC was calculated by subtracting basal OCR (point before adding first Glc) to CCCP induced OCR. (D) Agilent Seahorse XF based Cell Energy Phenotype Profile of SufT-KD. The relative utilization of the two energy pathways (oxidative phosphorylation and glycolysis) is determined under both baseline (Baseline Phenotype) and stressed (Stressed Phenotype) condition after CCCP addition. All point of OCR and ECAR are normalized to CFU (2x106 cells/well) and calculated in percent with respect to third basal point. Seahorse XF analyzer experiment was performed in triplicate with two independent replicates. Student’s t test (two tailed) was performed here with p values, *p≤0.05, **p≤0.01 and ***p≤0.001.
Fig 7.
SufT is essential for in vitro growth and regulates survival under redox stress and inside macrophages.
(A) A gradual decrease in the growth of the SufT-KD strain with increasing concentration of ATc (0–200 ng/mL) in standard liquid 7H9 medium. (B) Image of the serially diluted (N = undiluted) culture spotting of control as well as SufT-KD on 7H11 plates with and without ATc (200 ng/mL). Log phase culture of control +ATc, SufT-KD+ATc and–ATc were diluted to 0.15 OD (~2x 107 cells/mL) and treated with (C) H2O2 (0 mM, 5 mM and 10 mM) or (D) DETA NO (0 mM, 0.5 mM and 1 mM) for 12 h. Ex vivo survival of control +ATc, SufT-KD +ATc, and–ATc was performed in (E) THP-1 and (F) RAW264.7 cell line. Infection was performed at MOI: 2 for 4 h (0 h). CFU was measured at 0, 24, 48 and 72 h of infection. Experiments were performed in triplicate with two independent experiments. Two-way analysis of variance (ANOVA) as applicable with multiple comparisons test was employed to determine statistical significance. **p ≤ 0.01 and ***p ≤ 0.001.
Fig 8.
SufT is required in acute and chronic phase of infection in vivo.
(A) BALB/c mice (n = 6) were aerosol infected with SufT-KD strain at 200 CFU per lung at 0 day and divided into three groups of i) SufT-KD -DOX (no doxycycline treatment), ii) SufT-KD +DOX Acute (DOX started at 7 days after infection) and iii) SufT-KD +DOX Chronic (DOX started at 21 days after infection). Post infection six animals were sacrificed from each group on day 7, 21 and 42 and CFU load per lung was measured. (B) Gross pathology of lungs (upper panel) from SufT-KD -DOX and +DOX infected BALB/c mice was shown after 42 days of infection. White arrows indicate distinct granulomas. (C) Hematoxyline and eosin- stained lung sections after 42 days of infection. (D) Histo-pathological granuloma scoring of lung sections from–DOX and + DOX infected BALB/c mice. Student’s t test (two tailed) was performed to determine statistical significance as **p ≤ 0.01 and ***p≤0.001.