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

Abstract

Mycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-have a unique cell envelope critical for their survival and resistance. The cell envelope's assembly and maintenance influence permeability, making it a key target against multidrug-resistant strains. Disulfide bond (DSB) formation is crucial for the folding of cell envelope proteins. The DSB pathway in mycobacteria includes two enzymes, DsbA and VKOR, required for survival. Using bioinformatics and cysteine profiling proteomics, we identified cell envelope proteins dependent on DSBs. We validated via in vivo alkylation that key proteins like LamA (MmpS3), PstP, LpqW, and EmbB rely on DSBs for stability. Furthermore, chemical inhibition of VKOR results in phenotypes similar to those of Δvkor. Thus, targeting DsbA-VKOR systems could compromise both cell division and mycomembrane integrity. These findings emphasize the potential of DSB inhibition as a novel strategy to combat mycobacterial infections.

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.

Figure 1.

Lack of DSB formation causes growth and cell division defects in M. smegmatis. a, DsbA oxidizes secreted 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. 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 represents the average ±SD of at least two 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-e, Cell dimensions were measured from samples obtained from at least three independent experiments using FIJI (https://fiji.sc/). Data represents the average ±SD. Cell counts included: WT (n=495), WT++ (n=505), Δvkor++ (n=498), Δvkor+ (n=500). Statistical tests were done using Kruskal-Wallis multiple comparisons. p-values are depicted in GP style: ≤0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns).

Figure 2.

In silico and proteomic analyses reveal candidate DsbA substrates. a, Bioinformatic analysis to mine essential secreted genes identifies 19 candidate DsbA substrates (see Supplementary Table 1). 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. c, Volcano plot of protein abundance ratios of Δvkor+ compared to WT (see Supplementary Table 2). Dowregulated (green) or upregulated (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 Supplementary Table 4). d, Biotinylated-cysteine containing peptides were sorted by the presence of TM segments or signal sequences and their normalized abundance was plotted, data represents the median abundance. Statistical test was done using Kruskal-Wallis multiple comparisons of the mean. p-values are depicted in GP style: ≤0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). e, Normalized abundance of four proteins of interest detected by global proteomics are shown. Data represents the average ±SD of at least three independent replicas. f, Abundance of cysteine containing peptides of two proteins of interest detected by cysteine profiling proteomics. Data represents the average ±SD of at least three independent replicas.

Figure 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 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. Protein abundance was determined using α-RpoB as a loading control. Representative images are shown. Data represent average±SD of three independent experiments. b, MtLamA harbors one DSB. Top, multiple sequence alignment was done using clustal omega 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. Immunoblot is a representative image of at least three independent experiments. c, MtPstP harbors two DSBs. Similar as in b. d, MtLpqW harbors one DSB. Similar as in b. e, MtEmbB harbors two DSBs. Similar as in b.

Figure 4.

M. tuberculosis PstP harbors two essential consecutive DSBs. a, The first DSB between Cys359 and Cys380 of PstP provides more 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 diferentially alkylated as indicated in Fig. 2. 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 average±SD of three independent experiments. LOD: below the limit of detection. Cysteines are indicated in order: C189, C359, C380, C424, and C510. b, DSBs in PstP are required for survival. 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) in 7H9 broth supplemented with 400 nM aTc and 25 μM of acetamide (Ace), and incubated at 37°C for 24 h to enumerate bacteria. Statistical tests were done using one-way ANOVA multiple comparisons. p-values are depicted in GP style: ≤0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns).

Figure 5.

Targeting oxidative protein folding simultaneously affects multiple essential cell envelope proteins. a, M. smegmatis WT (with DMSO) or treated with 750 μM bromindione (BR) were grown in modified 7H9 broth at 37°C. Data represents the average ±SD of at least two independent experiments. b, M. smegmatis WT (with DMSO) or treated with 750 μM BR were 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 represents the average ±SD. Cell counts included: WT (n=495), WT+BR (n=490), Δvkor++ (n=498), Δvkor+ (n=500). Statistical tests were done using Kruskal-Wallis multiple comparisons. p-values are depicted in GP style: ≤0.0001 (****), 0.0002 (***), 0.021 (**), 0.0332 (*), and non-significant (ns). c, MtPstP is unstable when M. smegmatis is grown with a VKOR inhibitor (BR). M. smegmatis expressing FLAG-tagged proteins were grown with different concentrations of BR at 37°C for 36 h. 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. 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 Supplementary Table 4). Solid black arrows represent the flow of electrons and dotted black arrows represent the substrates of DsbA/VKOR pathway. MK-9: Menaquinone.