Fig 1.
The type II TA systems of mycobacteria were investigated. Schematic diagram of the toxin-antitoxin system.
(A) TA systems are annotated according to the GenBank database, excluding VapBC50 (rv3750c-rv3749c), VapBC49 (rv3180c-rv3181c), HigBA3 (rv3182-rv3183), HigBA2 (rv2022c-rv2021c), MazEF10 (rv0298-rv0299) and VapBC45 (rv2018-rv2019) systems; these systems are annotated according to Sala et al. [32]. The system RelBE3 (rv3358-rv3357, GenBank database, NCBI) is called the YefM/YoeB system by Sala. All of the TA systems depicted here are type II (systems marked with an asterisk are novel TA systems that are not classified to any family, but for which functional activity has been shown [32]). The 13 genes, our proposed set for genotyping, are highlighted in bold. (B) Type II TA systems are encoded by two genes, a toxin and an antitoxin, that form one operon with a promoter located upstream of the first antitoxin gene. PIN domain is the functional part of the toxin gene, the four conserved acidic residues marked at the picture: the three well-conserved acidic residues, at positions 4[D], 40[E] and 93[D], and with fourth acidic residue is less well conserved at position 112[D].
Table 1.
The division of Beijing genotype on subtypes using the polymorphisms in five genes belong to type II TA systems.
Table 2.
The minimum set of genes for genotyping strains of M. tuberculosis, developed on the basis of SNP in genes of TA systems of VapBC, HigAB and MazEF families.
Fig 2.
Phylogenetic relationship between different genotypes of the M. tuberculosis.
(A) Phylogenetic tree constructed on the basis of polymorphisms (SNP) in all of the considered genes of type II TA systems. An unrooted phylogenetic tree for the 173 strains from this study was constructed based on the presence/absence of SNPs in the nucleotide sequences of 71 TA systems (S3 Table); (B) Phylogenetic tree constructed on the basis of SNP in a minimum set of genes of type II TA systems. An unrooted phylogenetic tree for 173 strains constructed based on SNPs in the nucleotide sequences of 13 genes (Table 2). In both of cases strains included in the one cluster belong to the same genotype (various genotypes highlighted by color). The trees was constructed by the neighbor-joining approach. The TA systems sequences were retrieved from different databases (see Materials and Methods). Sequences were multiply aligned by using ClustalW ver. 2.1 software. The trees was calculated using MEGA ver. 6. Bootstrap support > 60% is indicated for the trees.
Fig 3.
Scheme of typing of M. tuberculosis strains using 13 genes of type II TA systems.
The algorithm for determining the genotype is presented. The scheme shows that, after the first iteration to determine the genotype, the number of genes for the analysis is decreased twofold. Each gene in the brackets is given its position that is replaced, and the appropriate nucleotide is indicated. All replacements are calculated relative to the reference strain H37Rv.
Table 3.
Determination of genotypes of tested strains in two ways.
Table 4.
Comparative genotyping of the Russian collection of isolates by two methods.
Fig 4.
Detection of the Ural genotype by qPCR.
Fluorescence in the FAM channel (blue): (1)13_2978, (2) 13–3114, (3) 13–3086, (4) 13_3158, (5) 13_4178, (6) 13_3539, (7) 13_2566, (8) 13_3632, (9) 13_3599, (10) 13_3896, (11) 13_3582, (12) 13_4189, (13) 13_3535, (15) 13_3147; Fluorescence in the HEX channel (green): (14) 13_3147, (16) 13_2978. Fluorescence of the channel FAM (blue) indicates the accumulation of the PCR product containing cytosine (C); the fluorescence of the channel HEX (green) indicates the accumulation of the PCR product containing thymine (T, the variable nucleotide) and indicates the SNP in the vapC10 gene (C394→T394) characteristic of the Ural genotype. Line 14 (13_3147) and 16 (13_2978) belong to the Ural genotype. For isolate 13_2978 fluorescence is detected on the two channels (FAM and HEX), this can indicate the presence of impurities (coinfection). qPCR fluorescence in RFU (relative fluorescence units) vs. PCR cycles. Intensity of fluorescence depending on the number of qPCR cycles for strains belonging to the Euro-American lineage.