Mejia-Santana Adrian, Collins Rebecca, Doud Emma H, Landeta Cristina
Department of Biology, Indiana University, , Bloomington, Indiana, USA.
Biochemistry and Molecular Biology, Indiana University School of Medicine, , Indianapolis, Indiana, USA.
mBio. 2025 Jul 31:e0108325. doi: 10.1128/mbio.01083-25.
Mycobacteria, including -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, iulfide bond formation protein (DsbA) and itamin epxide eductase (VKOR), for introducing disulfide bonds into exported proteins. 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 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.
分枝杆菌，包括结核病的病原体，具有独特且不可渗透的细胞壁，这对于其生存和抗生素耐药性至关重要。这种细胞壁的组装和维持依赖于正确折叠的蛋白质，然而二硫键形成在这些过程中的作用仍知之甚少。分枝杆菌依靠两种膜酶，二硫键形成蛋白（DsbA）和维生素环氧化物还原酶（VKOR），将二硫键引入输出蛋白中。研究预测，约64%的输出蛋白含有偶数个半胱氨酸残基，因此存在二硫键；然而，DsbA-VKOR途径的底物在很大程度上仍不清楚。在这里，我们证明DsbA和VKOR将二硫键引入底物蛋白，并鉴定了几种依赖于分枝杆菌细胞壁中氧化折叠的必需蛋白。使用生物信息学和半胱氨酸谱蛋白质组学，我们发现了许多需要二硫键来维持稳定性的输出蛋白。全细胞中的半胱氨酸衍生化证实，包括LamA（MmpS3）、PstP、LpqW和EmbB在内的关键蛋白依赖二硫键来发挥正常功能。此外，VKOR的化学抑制模拟了缺失的表型，从而突出了其在维持分枝杆菌细胞膜完整性中的重要作用。这些发现填补了理解分枝杆菌细胞壁生物合成方面的关键空白，并强调了DsbA-VKOR系统作为破坏耐药分枝杆菌细胞壁稳态的有前景靶点。
重要性
这项工作解决了理解分枝杆菌细胞壁过程中的一个主要缺陷，并突出了分枝杆菌中氧化蛋白折叠的生物学和临床意义。这个以二硫键形成为特征的过程对于输出蛋白的稳定性至关重要。虽然在革兰氏阴性菌中进行的二硫键形成研究表明在分枝杆菌中可能有类似作用，但二硫键的潜在影响仍不清楚。因此，我们开始使用生物信息学、蛋白质组学以及遗传和生化方法相结合的方式，研究分枝杆菌中依赖二硫键的多种生理功能。我们鉴定了数百种受氧化蛋白折叠影响的蛋白质，并验证了这一过程的必需底物。我们表明二硫键不仅对分枝杆菌关键蛋白的稳定性和功能至关重要，而且代表了一种对抗抗菌耐药性的新治疗靶点。我们的发现强调了靶向二硫键形成以破坏分枝杆菌细胞膜组装的潜力，为抗分枝杆菌药物开发开辟了新途径。
