Mycobacterium tuberculosis Type II Toxin-Antitoxin Systems: Genetic Polymorphisms and Functional Properties and the Possibility of Their Use for Genotyping

Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of Mycobacterium tuberculosis in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of M. tuberculosis was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of M. tuberculosis strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of M. tuberculosis. The possibility of using our set of genes for genotyping using PCR is also demonstrated.目前，IS元件（IS-elements）、DR元件（DR-elements）、可变数目串联重复序列（variable number tandem repeats, VNTR）、持家基因及其他基因家族中的单核苷酸多态性（single nucleotide polymorphisms, SNPs）等多种遗传标记，均被应用于基因分型研究。本研究提出一种全新的分型思路，即采用II型毒素-抗毒素（toxin-antitoxin, TA）系统作为标记基因。II型TA系统在致病性、耐受性及持留表型的形成过程中发挥关键作用，进而影响结核分枝杆菌（Mycobacterium tuberculosis）在宿主不同发育阶段（定植、巨噬细胞感染等）的存活能力。绝大多数TA系统基因协同行使功能，形成统一的调控网络：某一对TA系统的抗毒素可与其他TA对甚至其他TA家族的毒素发生相互作用。本研究对173株已完成全基因组测序的结核分枝杆菌的II型TA系统基因开展了生物信息学分析，发现部分II型TA系统基因携带与特定基因型相关的单核苷酸多态性位点。我们提出一套最小化核心TA系统基因集，可用于将结核分枝杆菌菌株划分为9种基础基因型，并可进一步细分亚型。利用该基因集，我们对包含62株结核分枝杆菌临床分离株的菌株集合完成了基因分型，并验证了该基因集可通过聚合酶链式反应（PCR）实现结核分枝杆菌基因分型的可行性。