Damiano Deborah K, Azevedo Bruna O P, Fernandes George S C, Teixeira Aline F, Gonçalves Viviane M, Nascimento Ana L T O, Lopes Alexandre P Y
Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo 05503-900, Brazil.
Programa de Pós-Graduação Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Prof. Lineu Prestes, 1730, São Paulo 05508-900, Brazil.
Microorganisms. 2024 Aug 13;12(8):1660. doi: 10.3390/microorganisms12081660.
Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene in did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of co-expressing genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from . Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium.
细菌普遍存在的毒素-抗毒素（TA）系统被认为是应激条件下重要的生存机制。在正常环境条件下，抗毒素会阻断毒素的作用，而在失衡状态下，抗毒素浓度降低，使细菌细胞暴露于一系列毒性事件中。这种失衡最明显的后果是细胞生长停滞，这就是TA系统通常被描述为在细菌生长动力学中起作用的原因。毒力相关蛋白B和C（VapBC）是II型TA系统家族，其中VapC预计具有毒性核糖核酸酶活性，而VapB可抵消这种活性。此前，我们利用计算机数据在巴西人类钩端螺旋体病的主要病原体哥本哈根血清型中确定了四个VapBC TA模块。本研究旨在获得这些蛋白质并对VapBC-1模块进行功能表征。毒素基因在肉汤培养中的表达并未降低细胞生长速率，而在活跃的TA模块中预期会出现这种情况。然而，有趣的是，当将毒素的表达与复合毒素和抗毒素的表达进行比较时，细胞活力受到强烈影响，菌落形成单位（CFU）下降了三个数量级。通过观察共表达基因的培养物中共纯化的毒素和抗毒素，体内证实了毒素与抗毒素之间的亲和力。核糖核酸酶活性测定表明，VapC-1可切割来自的MS2 RNA和核糖体RNA。我们的结果表明，VapBC-1模块是一个潜在的功能性TA系统，作用于涉及特定功能的靶标。必须强调的是，不能仅仅基于TA模块在液体培养基中抑制细菌生长的能力来定义其功能的常见归属。
