Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis

Abstract

Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe-host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn²⁺) under oxidative stress. Next, we identified a T6SS-4-dependent Mn²⁺-binding effector TseM, and its interacting partner MnoT, a Mn²⁺-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn²⁺ conditions. Both TseM and MnoT are required for efficient uptake of Mn²⁺ across the outer membrane under Mn²⁺-limited and -oxidative stress conditions. The TseM-MnoT-mediated active Mn²⁺ transport system is also involved in contact-independent bacteria-bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria-bacteria competition.

Keywords: Burkholderia; ion uptake; outer membrane transporter; oxidative stress; type VI secretion.

Fig. 1.

OxyR-regulated T6SS-4 is involved in oxidative stress resistance. (A) Genes differentially transcribed in the B. thailandensis oxyR mutant compared with those in the wild type were detected by transcriptomic and qRT-PCR analyses. Fourteen representative genes were chosen to validate the RNA-seq data by qRT-PCR. The white bars represent the mean values obtained for the reference wild type and three biological replicates. Error bars indicate the SD. Black bars represent RNA-seq data. (B) Binding of OxyR to the T6SS-4 promoter. Interaction of OxyR with a biotin-labeled probe was detected using streptavidin-conjugated HRP and a chemiluminescent substrate. An unlabeled promoter was added to determine the binding specificity of OxyR. Bio-P_T6SS-4, biotin-labeled T6SS-4 promoter. (C and D) The indicated strains grown to the stationary phase were exposed to diverse stress for 40 min and the viability of the cells was determined. Data shown are the average and SD from three independent experiments. (E) Deletion of T6SS-4 led to accumulation of intracellular ROS under oxidative conditions. Intracellular ROS in stationary phase bacterial strains exposed to CHP was determined with the H₂DCFDA probe. Data shown are the average and SD from three independent experiments. (F) Oxidative stress induced the expression of T6SS-4. B. thailandensis strains were treated with the indicated amounts of CHP and the expressions of the major components of T6SS-4 were measured by qRT-PCR. Data shown are the average and SD from three independent experiments. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.

Fig. S1.

OxyR regulates the expression of T6SS-4. (A) The ΔoxyR mutant was highly resistant to oxidative stress. The survival rate was measured by viability assay. Mean values with SDs (error bars) from at least three repeats are shown. *P < 0.05. (B) Growth curves of the wild-type (WT), ΔoxyR mutant, and the complemented strain ΔoxyR(oxyR) under normal condition. The growth of the indicated strains in LB was monitored by measuring OD₆₀₀ at indicated time points. (C) The levels of hcp4 mRNAs in exponentially growing B. thailandensis cells with (+) or without (−) 20-min exposure to 0.15 mM CHP was determined by quantitative RT-PCR. The mRNA levels are presented relative to the value obtained from WT cells without CHP treatment. (D) The protein level of Hcp4 in differently treated WT and ΔoxyR mutant strains. Lysates from bacteria with or without (control) 30-min exposure to 0.15 mM CHP were resolved by SDS/PAGE, and Hcp4 was detected by immunoblotting using a specific anti-Hcp4 antibody. For the pellet fraction, isocitrate dehydrogenase (ICDH) was used as a loading control. (E) Identification of the OxyR binding site in the promoter region of T6SS-4. Putative OxyR binding site identified by the online software Virtual Footprint (www.prodoric.de/vfp) was indicated by shading. The ATG start codon of the first ORF of the T6SS-4 operon was marked in boldface, and the –35 and –10 elements of the T6SS-4 promoter are boxed. +1 denotes the transcription start point.

Fig. 2.

T6SS-4 is important for the accumulation of intracellular Mn²⁺ under oxidative stress conditions. (A) The alleviation of the sensitivity of B. thailandensis strains to CHP by exogenous Mn²⁺ (0.25 μM) required T6SS-4. Relevant stationary phase bacterial strains were exposed to 0.25 mM CHP in M9 medium with or without exogenously provided Mn²⁺ (0.25 μM) for 40 min and the viability of the cells was determined. The mean values and SDs from at least three repeats are shown. (B) Reduction of intracellular ROS in CHP-treated B. thailandensis strains by exogenous Mn²⁺ (0.25 μM) required T6SS-4. The mean values and SD from at least three repeats are shown. (C) Mn²⁺ uptake required T6SS-4 under oxidative stress conditions. Stationary phase B. thailandensis strains were exposed to 0.25 mM CHP for 20 min in PBS containing 0.25 μM MnSO₄. Mn²⁺ associated with bacterial cells was measured by inductively coupled plasmon resonance atomic absorption spectrometry (ICP-MS). (D) T6SS-4 expression is inhibited under high Mn²⁺ conditions. B. thailandensis wild type was grown in LB containing 10 or 100 μM Mn²⁺, and the expression of the major T6SS-4 genes was measured by qRT-PCR. Data shown are the average of three independent experiments and error bars indicate the SD from three independent experiments. **P ≤ 0.01; *P ≤ 0.05.

Fig. S2.

T6SS-4 and MnoT are required for optimal growth under oxidative stress. Saturated bacterial cultures were diluted to an OD₆₀₀ of 0.05 in LB medium (A and C); LB medium with 250 μM EDTA (A and C); LB medium with 240 μM CHP and 250 μM EDTA (B and D); and LB medium with 240 μM CHP, 250 μM EDTA, and 250 μM Mn²⁺ (B and D). The growth of the cultures was monitored by measuring OD₆₀₀ at indicated time points. Data shown were the average of three independent experiments; error bars indicate SD from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 3.

A Mn²⁺-binding protein translocated by T6SS-4 resisted oxidative stress. (A) TseM is a secreted substrate of T6SS-4. Proteins in culture supernatant of the relevant B. thailandensis strains were probed using specific anti-TseM antibody. For the pellet fraction, isocitrate dehydrogenase (ICDH) was used as a loading control. (B) The binding of divalent ions by TseM was detected by atomic absorption spectrometry. (C) The binding of Mn²⁺ by TseM. Mn²⁺-free TseM (Upper) or TseM* (TseM^{Q35R/H63A/N132R}) (Lower) was used to evaluate Mn²⁺-binding activity by isothermal titration calorimetry (ITC). Data were analyzed using the NanoAnalyze software (TA Instruments). (D) The alleviation of the sensitivity of B. thailandensis strains to CHP by exogenous Mn²⁺ (0.25 μM) required the TseM protein. The viability of stationary phase B. thailandensis strains was determined after exposure to CHP, or CHP and 0.25 μM Mn²⁺ for 40 min. (E) Deletion of the tseM gene led to an accumulation of intracellular ROS. The intracellular levels of ROS were determined with the H₂DCFDA probe after stationary phase B. thailandensis strains were exposed to CHP, or CHP with 0.25 μM Mn²⁺ for 40 min. (F) TseM is involved in Mn²⁺ acquisition. Stationary phase B. thailandensis strains were exposed to 0.25 mM CHP for 20 min in PBS containing 0.25 μM MnSO₄. Mn²⁺ associated with bacterial cells was determined by ICP-MS. (G) TseM expression is inhibited by high Mn²⁺ conditions. B. thailandensis wild-type cells were grown in LB medium with 10 and 100 μM Mn²⁺, and the expression of tseM was measured using qRT-PCR. (H) The rescue of the tseM or clpV4 mutant using recombinant TseM protein. Recombinant TseM or TseM* (TseM^{Q35R/H63A/N132R}) protein at 1 μM was added to bacterial survival experiments in M9 medium before viability assessment. Mutants complemented with the corresponding gene were used as controls. The mean values and SDs from at least three repeats are shown. **P ≤ 0.01; *P ≤ 0.05; n.s., not significant.

Fig. S3.

Identification of a putative T6SS effector in the B. thailandensis T6SS-4 gene cluster. (A) The structure of B. thailandensis T6SS-4 gene cluster is similar to the T6SS-4 gene cluster from Y. pseudotuberculosis. The tseM gene (bth_II1883, indicated in red) localizes in the end of the T6SS-4 operon. (B) Phylogenetic relationship of B. thailandensis TseM with homologous proteins in other relative Burkholderia. Different protein sequences were obtained from the SwissProt database. The phylogenetic tree was constructed using MEGA 6.0 by the neighbor-joining method and multiple sequence alignment was performed using CLUSTAL W. The scale bar indicates percentage of divergence (distance). Accession numbers are as follows: B. thailandensis E264 (ABC35934); B. thailandensis E254 (AIT22423); B. thailandensis (AIP65980); B. thailandensis USAMRU Malaysia (AIC89753); B. thailandensis MSMB121 (AGK49752); B. mallei (AIO54172); B. mallei SAVP1 (ABM48775); B. mallei NCTC 10247 (AIS27138); B. pseudomallei 668 (ABN88025); B. pseudomallei A79A (AIV92858); B. pseudomallei K96243 (YP_110555); B. pseudomallei NAU35A-3 (AIS91265); B. oklahomensis C6786 (AJX34588); Burkholderia sp. 2002721687 (AJY39125); Burkholderia sp. Bp5365 (ALX46218); Celeribacter marinus IMCC12053_1024 (ALI54972.1); and Rhizobium tropici CIAT 899 (AGB70070.1). (C) Side and top 3D model of TseM-ion binding built with phyre². The ion is illustrated as a yellow sphere.

Fig. S4.

Effects of different clpVs and vgrGs on TseM secretion. (A) Secretion of TseM in the Δ4clpV mutant complemented with clpV1, clpV2, clpV4, and clpV6, respectively. (B) Secretion of TseM in the ΔvgrG4a4b mutant complemented with vgrG4a and vgrG4b, respectively. (C) Secretion of TseM in the ΔicmF4 mutant and the complemented strain. Proteins in culture supernatant were probed using specific anti-TseM antibody. For the pellet fraction, isocitrate dehydrogenase (ICDH) was used as a loading control.

Fig. S5.

TseM and MnoT are not involved in iron and zinc accumulation. (A) TseM (BTH_II1883) does not bind iron. Iron binding analysis by 15% native PAGE. Lanes 1 and 2 show the gel stained with Coomassie bright blue. Lanes 3 and 4 show the same gel stained for iron by the potassium ferricynaide method. His₆-Fur was used as a positive control. Lanes 1 and 3 show TseM (BTH_II1883). Lanes 2 and 4 show His₆-Fur. (B) TseM (BTH_II1883) does not bind Zn²⁺. Spectral scans of solutions containing 10 µM PAR without Zn²⁺ (black), with Zn²⁺ (red), or with Zn²⁺ and increasing concentrations of recombinant TseM (BTH_II1883) and control buffer (different color) are shown. (C) MnoT is not involved in iron and zinc accumulation in B. thailandensis. Stationary phase B. thailandensis strains were exposed to 0.25 mM CHP for 20 min in PBS containing 1 μM FeCl₃ or 1 μM ZnSO₄. Iron and zinc ions associated with bacterial cells were measured using ICP-MS. Data shown are the average and SD from three independent experiments. n.s., not significant.

Fig. S6.

Effects of Mn²⁺ and CHP on T6SS-4 and mnoT expression. (A) B. thailandensis wild type was grown in M9 medium containing different concentrations of Mn²⁺ with or without CHP (100 µM ), and the expression of clpV4, hcp4, icmF4, and tseM was measured by qRT-PCR. (B) The expression of mnoT is regulated by Mn²⁺. B. thailandensis wild type was grown in M9 medium containing different concentrations of Mn²⁺, and the expression of mnoT was measured by qRT-PCR. Data shown were the average of three independent experiments; error bars indicate SD from three independent experiments. *P < 0.05.

Fig. 4.

TseM interacts with a TBDT family transporter involved in Mn²⁺ transport. (A) MnoT was retained by agarose beads coated with GST-TseM. GST-Bind beads coated with GST-TseM (lanes 2, 3, and 5) or GST (lanes 1 and 4) were incubated with CHP-treated B. thailandensis supernatant (lanes 1 and 2) or cell lysates (lanes 4 and 5). After washing with PBS, the proteins resolved by SDS/PAGE were visualized using silver staining, and bands that specifically retained by the GST-TseM–coated beads were identified by mass spectrometry. (B) Direct binding of TseM to MnoT. His₆-MnoT was incubated with GST-TseM or GST, and the protein complexes captured with glutathione beads were detected by Western blotting. (C) MnoT is involved in Mn²⁺ acquisition in B. thailandensis. Stationary phase B. thailandensis strains were exposed to 0.25 mM CHP for 20 min in PBS containing 0.25 μM MnSO₄. Mn²⁺ associated with bacterial cells was determined by ICP-MS. Data shown are the average and SD from three independent experiments. (D) MnoT expression was inhibited by high Mn²⁺ conditions and induced by CHP and EDTA. Cells of B. thailandensis wild type were grown in LB medium with 100 μM Mn²⁺, 100 μM CHP, 100 μM EDTA, or 100 μM EDTA together with 100 μM Mn²⁺ (EDTA + 1× Mn²⁺), and 100 μM EDTA together with 200 μM Mn²⁺ (EDTA + 2× Mn²⁺). The expression of mnoT was measured by qRT-PCR. Data shown are the average and SD from three independent experiments. **P ≤ 0.01; *P ≤ 0.05. (E) The expression of MnoT was negatively regulated by OxyR. Cells of relevant B. thailandensis strains were grown in LB medium and the expression of mnoT was measured by qRT-PCR. Data shown are the average and SD from three independent experiments. (F) Alleviation of the sensitivity of B. thailandensis strains to CHP by exogenous Mn²⁺ (0.25 μM) required MnoT. The viability of relevant stationary B. thailandensis strains was determined after exposure to CHP or CHP with 0.25 μM Mn²⁺ for 40 min. The mean values and SD from at least three repeats are shown. **P ≤ 0.01; *P ≤ 0.05.

Fig. S7.

Sequence analysis of MnoT. (A) Pertinent secondary structure elements of MnoT. Plug, the TonB-plug domain (residues 68–181); TonB-dep-Rec, the TonB-dependent receptor domain (residues 529–777). (B) Phylogenetic relationship of TonB-dependent outer membrane receptors. Different protein sequences were obtained from the SwissProt database. The phylogenetic tree was constructed using MEGA 6.0 by the neighbor-joining method and multiple sequence alignment was performed using CLUSTAL W. The scale bar indicates percentage of divergence (distance). SwissProt accession nos. of proteins from species are as follows: B. thailandensis BTH_I1598 (gi:83652520); N. meningitidis ZnuD (gi:15676857); Neisseria gonorrheae TonB-dependent receptor protein (gi:59801564); Mannheimia haemolytica iron-regulated outer membrane protein (gi:13591379); Moraxella catarrhalis HumA (gi:53829495); Haemophilus parasuis TonB-dependent receptor (gi:219870611); Actinobacillus pleuropneumoniae iron-regulated outer membrane protein (gi:165976339); Bordetella pertussis outer membrane protein (gi:33594004); Burkholderia cepacia TonB-dependent receptor (gi:402248466); Salmonella enterica FepA (gi:228960703); Pseudomonas aeruginosa FepA (gi:15597884); Pseudomonas aeruginosa PhuR (gi:15599904); Sinorhizobium meliloti ShmR (gi:470189354); Y. pseudotuberculosis HasR (gi:169752827); Beauveria bassiana TonB-dependent heme receptor A (gi:701779958); Haemophilus influenzae hemin receptor (gi:16272087); Y. pseudotuberculosis HemR (gi:170026102); E. coli heme utilization/transport protein (gi:15833634); Helicobacter pylori FrpB4 (gi:15646121); Ancylostoma ceylanicum TonB-dependent receptor (gi:510848345); Shewanella frigidimarina TonB-dependent receptor (gi:114335087); and Riemerella anatipestifer TonB-dependent receptor (gi:312446409).

Fig. 5.

The Mn²⁺ transport activity of TseM is dependent on MnoT. (A) The indicated bacterial strains grown to the stationary phase were exposed to CHP (0.25 mM) for 40 min in M9 medium containing 25 nM Mn²⁺, and the viability of the cells was determined. (B) Intrabacterial growth competition assays between the indicated competitor strains (x-axis) following incubation with participant strains at 37 °C for 12 h in M9 medium containing 20 μM CHP. The competitive index result is calculated as the final cfu ratio (competitor/participant) divided by the initial ratio. (C) Interbacterial growth competition assays between B. thailandensis and E. coli K12. Quantification of cfu before (initial) and after (final) growth competition assays between the indicated organisms. The cfu ratio of the relevant B. thailandensis strains versus the competitors is plotted. (D) MnoT enhanced Mn²⁺ accumulation in E. coli K12. Stationary phase E. coli K12(pME6032) and E. coli K12(pME6032-mnoT) strains were cultivated in M9 medium containing 50 μM CHP, 25 nM Mn²⁺, with (+) or without (−) 1 μM TseM, and intracellular Mn²⁺ was measured by ICP-MS. (E) Virulence survival of relative B. thailandensis strains in G. mellonella larvae. Ordinate represented the mean percentage survival rate of G. mellonella infected with different strains after 16 h. Error bars represent the SD from three independent experiments. **P ≤ 0.01; *P ≤ 0.05.

Fig. S8.

MnoT homologs are widely distributed in Burkholderia species. The phylogenetic tree generated by the minimum-evolution algorithm in MEGA 6.0 illustrates that MnoT is highly conserved among the vast majority of Burkholderia. The B. thailandensis E264 MnoT was indicated in red. The bar represents the genetic distance.

Fig. 6.

Model of T6SS-4–mediated Mn²⁺ transport and oxidative resistance in B. thailandensis.