Disulfide bonds are critical for stabilizing cell division, cell envelope biogenesis, and antibiotic resistance proteins in mycobacteria

Abstract

Mycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-possess a unique and impermeable cell envelope that is critical for survival and antibiotic resistance. The assembly and maintenance of this envelope depend on properly folded proteins, yet the role of disulfide bond formation in these processes remains poorly understood. Mycobacteria rely on two membrane enzymes, disulfide bond formation protein A (DsbA) and vitamin K epoxide reductase (VKOR), for introducing disulfide bonds into exported proteins. In silico studies predict that ~64% of exported proteins contain even numbers of cysteine residues and thence disulfide bonding; nevertheless, substrates of the DsbA-VKOR pathway remain largely unknown. Here, we demonstrate that DsbA and VKOR introduce disulfide bonds into substrate proteins and identify several essential proteins that depend on oxidative folding in the mycobacterial cell envelope. Using bioinformatics and cysteine profiling proteomics, we uncover numerous exported proteins that require disulfide bonds for stability. Cysteine derivatization in whole cells confirms that key proteins, including LamA (MmpS3), PstP, LpqW, and EmbB, rely on disulfide bonds for proper function. Furthermore, chemical inhibition of VKOR phenocopies vkor deletion, thus highlighting its essential role in maintaining mycomembrane integrity. These findings address a critical gap in understanding mycobacterial cell envelope biogenesis and underscore the DsbA-VKOR system as a promising target for disrupting cell envelope homeostasis in drug-resistant Mycobacteria.IMPORTANCEThis work addresses a major deficiency in understanding mycobacterial cell envelope processes and highlights the biological and clinical implications of oxidative protein folding in mycobacteria. This process, marked by the formation of disulfide bonds, is essential for the stability of exported proteins. While disulfide bond formation studies in Gram-negative bacteria suggested a similar role in mycobacteria, the underlying consequences of disulfide bonds remained unclear. Thus, we began investigating the diverse physiological functions dependent on disulfide bonds in Mycobacteria using a combination of bioinformatics, proteomics, and genetic and biochemical approaches. We identified hundreds of proteins affected by oxidative protein folding and validated essential substrates of this process. We show that disulfide bonds are not only crucial for the stability and function of key mycobacterial proteins but also represent a novel therapeutic target against antimicrobial resistance. Our findings underscore the potential of targeting disulfide bond formation to disrupt mycomembrane assembly, opening new avenues for antimycobacterial drug development.

Keywords: AftB; AftD; DsbA; EccB3; EmbB; LamA; LpqW; MmpS3; MycP3; PP2C; PstP; Rv2507; Ser/Thr phosphatase; VKOR; actinobacteria; disulfide bonds; essential proteins; mycobacteria; mycomembrane; oxidative protein folding; substrates.

Fig 1

Lack of DSB formation causes growth and cell division defects in M. smegmatis. (a) DsbA oxidizes exported proteins and becomes reduced. VKOR regenerates reduced DsbA by transferring the electrons to menaquinone. Black arrows represent the flow of electrons, IM: inner membrane, MM: mycomembrane, MK-9: menaquinone-9. The schematic figure was created in BioRender. (b) M. smegmatis ΔdsbA and Δvkor survival is affected in modified 7H9 broth. ΔdsbA was supplemented with 100 nM of aTc to induce MsdsbA expression, and Δvkor was supplemented with 1 mM cystine (CSSC). Data represent the average ±SD of at least three independent experiments. (c) WT and Δvkor cells, supplemented with 1 mM (++) or 100 µM (+) cystine, were fluorescently stained with 50 nM Syto24 to stain nucleic acids and 0.6 µg/mL FM4-64 to stain the membrane. Representative images of the phenotypes are shown. (d and e) Cell dimensions were measured from samples obtained from at least three independent experiments using FIJI (https://fiji.sc/). Data represent the average ±SD. Cell counts included: WT (n = 495), WT++ (n = 505), Δvkor++ (n = 498), Δvkor+ (n = 500). Statistical tests were done using the Kruskal-Wallis multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns).

Fig 2

In silico and proteomic analyses reveal candidate DsbA substrates. (a) Bioinformatic analysis to mine essential exported genes identifies 19 candidate DsbA substrates (see Table S1). (b) Global and cysteine profiling proteomics approach to identify potential DsbA substrates (see text for further details). TCEP, tris(2-carboxyethyl)phosphine; IAM, iodoacetamide; DBIA, desthiobiotin-iodoacetamide; TMT, tandem mass tag; MS, mass spectrometry. The schematic figure was done with BioRender. (c) Volcano plot of protein abundance ratios of Δvkor+ compared to WT (see Table S2). Decreased (green) or increased (pink) proteins with log2 ratios of ±1 and P-value ≤ 0.05 are highlighted. Red dots indicate proteins analyzed or discussed in this work (see Table S5), yellow dots indicate proteins for which cysteines were enriched and exported, and orange dots indicate essential proteins that overlap with in silico and proteomics approach (see Table S3). (d) Biotinylated-cysteine-containing peptides were sorted by the presence of TM segments or signal sequences and their normalized abundance was plotted. The data represent the median abundance. A statistical test was done using the Kruskal-Wallis multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). (e) The normalized abundance of four proteins of interest was detected by global proteomics. Data represent the average ±SD of at least three independent replicas. Statistical test was done using two-way ANOVA and Sidák’s multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). (f) Abundance of cysteine-containing peptides of two proteins of interest was detected by cysteine profiling proteomics. Data represent the average ±SD of at least three independent replicas. Statistical test was done using two-way ANOVA and Sidák’s multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns).

Fig 3

M. tuberculosis LamA, PstP, LpqW, and EmbB are substrates of DsbA/VKOR. (a) MtLamA, MtPstP, MtLpqW, and MtEmbB require DSBs for stability. M. tuberculosis proteins were fused to a 3X-FLAG tag at their carboxy termini and expressed in M. smegmatis WT and Δvkor supplemented with 1 mM (++) or 0.4–0.5 mM cystine (+). Cells were grown at 37°C in the presence of 2.5 nM (EmbB) or 200 nM aTc for 36 h (PstP and LpqW), or 200 nM aTc for 18 h (LamA). Proteins were precipitated from cell lysates, quantified, and reduced before being separated by SDS-PAGE. Representative images are shown. (b) Protein abundance was determined by band density using α-RpoB as a loading control. Data represent average ±SD of three independent experiments. A statistical test was done using two-way ANOVA and Dunnett’s multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). (c) In vivo differential alkylation diagram (see text for further details). DTT, dithiothreitol; NEM, N-ethylmaleimide; MalPEG, α-[3-(3-Maleimido-1-oxopropyl)amino]propyl-ω-methoxy, polyoxyethylene, 2-5 kDa. (d) MtLamA harbors one DSB. Top, multiple sequence alignment was done using Clustal Omega (48) and Jalview to visualize it (https://www.jalview.org/). Bottom, M. smegmatis WT and Δvkor expressing MtLamA were grown and induced as indicated in a. Experimental protein samples (indicated with a bracket) were differentially alkylated by treating them with 20 mM NEM to block free thiols. Disulfide-bonded cysteines were then reduced with 100 mM DTT, and new thiols were alkylated with 12.5 mM MalPEG2k. Controls were treated with 100 mM DTT and then alkylated with either 20 mM NEM or 12.5 mM MalPEG2k. Δvkor samples were loaded in excess to be able to observe alkylated bands. Western blotting using α-FLAG antibody was used to detect LamA. The immunoblot is a representative image of at least three independent experiments. (e) MtPstP harbors two DSBs. Similar to d. (f) MtLpqW harbors one DSB. Similar to d. (g) MtEmbB harbors two DSBs. Similar to d.

Fig 4

M. tuberculosis PstP harbors two essential consecutive DSBs. (a) The first DSB between Cys359 and Cys380 of PstP provides more protein stability than the DSB between Cys424 and Cys510. Cells were grown at 37°C for 36 h in the presence of 200 nM aTc. Proteins were precipitated, reduced with 100 mM DTT, and alkylated with 12.5 mM MalPEG2k. Controls were either reduced or differentially alkylated as indicated in Fig. 3. Immunoblot is a representative image of three independent experiments. Protein abundance was determined using reduced samples, and α-RpoB was used as a loading control. Data represent the average ±SD of three independent experiments. LOD: below the limit of detection. Cysteines are indicated in order: C189, C359, C380, C424, and C510. C, cytoplasm, P, periplasm. The schematic figure was created in BioRender. (b) DSBs in PstP are required for normal growth. M. smegmatis pstP was silenced using CRISPRi, while an ectopic copy of M. tuberculosis pstP, either WT or cysteine mutants, was used to rescue the knockdown growth. Cells were inoculated to an OD₆₀₀ of 0.01 (CFU/mL indicated as red dotted line, see Source Data) in 7H9 broth supplemented with 400 nM anhydrotetracycline (aTc) and 25 µM of acetamide (Ace), and incubated at 37°C for 24 h to enumerate bacteria. Statistical tests were done using a one-way ANOVA multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). The schematic figure was created in BioRender.

Fig 5

Targeting oxidative protein folding simultaneously affects multiple essential cell envelope proteins. (a) M. smegmatis WT treated with DMSO or 750 µM bromindione (BR) was grown in modified 7H9 broth at 37°C. Data represent the average ±SD of at least three independent experiments. (b) M. smegmatis WT treated with DMSO or 750 µM BR was grown in modified 7H9 broth at 37°C for 36 h. Cells were fluorescently stained with 50 nM Syto24 to stain nucleic acids and 0.6 µg/mL FM4-64 to stain the membrane. Cell dimensions were measured using FIJI (https://fiji.sc/). Data represent the average ±SD. Cell counts included: WT (n = 495), WT + BR (n = 490), Δvkor++ (n = 498), and Δvkor+ (n = 500). Statistical tests were done using the Kruskal-Wallis multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). (c) Essential proteins are unstable when M. smegmatis is grown with a VKOR inhibitor (BR). M. smegmatis expressing FLAG-tagged proteins was grown with different concentrations of BR at 37°C for 24 (MtLamA) or 36 h (MtPstP and MtLpqW). Proteins were precipitated from cell lysates and reduced before being separated by SDS-PAGE. Protein abundance was determined using α-RpoB as a loading control. Values represent the average ±SD of three independent experiments. Statistical tests were done using one-way ANOVA and Dunnett’s multiple comparisons test. P-values are depicted in GP style: ≤ 0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). (d) DSBs are present in proteins involved in M. tuberculosis cell envelope biogenesis and cell division (see text for further details). Proteins in red text were experimentally demonstrated to have DSBs in this work, while proteins in blue text are predicted substrates identified in this work and structural studies (see Table S5). Solid black arrows represent the flow of electrons, and dotted black arrows indicate the substrates of the DsbA/VKOR pathway. MK-9, menaquinone; PIM, phosphatidylinositol mannosides; LM, lipomannan, LAM, lipoarabinomannan; a, acyl-carrier protein; ESX-3, specialized secretion system. The schematic figure was created in BioRender.