VapC Toxins from Mycobacterium tuberculosis Are Ribonucleases that Differentially Inhibit Growth and Are Neutralized by Cognate VapB Antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的染色体编码47个属于VapBC家族的毒素-抗毒素模块。这类模块在结核分枝杆菌生理过程中的作用，以及其扩张所承担的功能目前仍未明确。本研究针对结核分枝杆菌的10个vapBC模块以及耻垢分枝杆菌（M. smegmatis）的单个vapBC模块展开了探究。在本次评估的结核分枝杆菌vapC基因中，仅Rv0549c、Rv0595c、Rv2549c和Rv2829c在通过四环素调控启动子在耻垢分枝杆菌中表达时表现出毒性。上述基因在结核分枝杆菌中条件性表达时同样显现出毒性。Rv2549c在耻垢分枝杆菌中的毒性与蛋白表达水平呈正相关，这表明VapC的浓度必须达到阈值方可观测到毒性效应。此外，在耻垢分枝杆菌中诱导产生的Rv2456蛋白水平显著低于Rv2549c，这或许可以解释该蛋白以及其他被归类为“无毒性”的VapC为何未表现出毒性。毒性VapC所介导的生长抑制效应，可通过共表达其同源VapB（作为vapBC操纵子的一部分或从其他染色体位点表达）得以中和，而异源抗毒素则无此类中和效果。上述结果证实了VapC与其同源VapB之间存在特异性相互作用，酵母双杂交（yeast two-hybrid）分析也验证了这一发现。对选定的vapC或vapBC基因进行敲除并不会影响分枝杆菌的体外生长，但会使菌体在毒性VapC表达后更易受到生长抑制。然而，即便敲除了对应的同源vapB基因，“无毒性”VapC的毒性也未在敲除突变菌株中显现，推测这可能是由于VapC蛋白水平不足所致。结合已被证实具有核糖核酸酶（ribonuclease, RNase）活性的Rv0065和Rv0617——这两种VapC蛋白分别与Rv0549c和Rv3320c具有序列同源性——的相关研究结果，本研究提示VapBC家族可能为结核分枝杆菌提供了丰富的核糖核酸酶活性来源，这或许会对该菌体的生理过程产生深远影响。