The type II TA systems of mycobacteria were investigated. Schematic diagram of the toxin-antitoxin system.

<p>(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. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]. 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]). 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].</p

Detection of the Ural genotype by qPCR.

<p>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 <i>vapC10</i> 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.</p

Scheme of typing of <i>M</i>. <i>tuberculosis</i> strains using 13 genes of type II TA systems.

<p>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.</p

Phylogenetic relationship between different genotypes of the <i>M</i>. <i>tuberculosis</i>.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.s003" target="_blank">S3 Table</a>); (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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.t002" target="_blank">Table 2</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#sec002" target="_blank">Materials and Methods</a>). 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.</p

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

<div><p>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 <i>Mycobacterium tuberculosis</i> 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 <i>M</i>. <i>tuberculosis</i> 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 <i>M</i>. <i>tuberculosis</i> 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 <i>M</i>. <i>tuberculosis</i>. The possibility of using our set of genes for genotyping using PCR is also demonstrated.</p></div

Comparative Genomic Analysis of <em>Mycobacterium tuberculosis</em> Drug Resistant Strains from Russia

<div><p>Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) <i>Mycobacterium tuberculosis</i> (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes – drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998–1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all <i>M. tuberculosis</i> genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4^XDR, belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4^XDR may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.</p> </div

Description of <i>M. tuberculosis</i> clinical isolates involved in this study.

1<p>RIF - rifampicin, INH - isoniazid, EMB - ethambutol, STR - streptomycin, PZA - pyrazinamide, ETH - ethionamide, AMI- amikacin, CAPR - capreomycin, OFL - ofloxacin.</p>2<p>according PHRI TB Centre database.</p>3<p>SpolDB4 was used for identification of data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056577#pone.0056577-Brudey1" target="_blank">[54]</a>.</p>4<p>24– VNTR: MIRU 02, MIRU 04, MIRU 10, MIRU 16, MIRU 20, MIRU 23, MIRU 24, MIRU 26, MIRU 27, MIRU 31, MIRU 39, MIRU 40, VNTR 42, VNTR 43, VNTR 1955, QUB-11b, ETRA, VNTR 46, VNTR 47, VNTR 48, VNTR 49, VNTR 3690, QUB-26, VNTR 53 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056577#pone.0056577-Supply1" target="_blank">[29]</a>.</p>5<p>Ural-1 subfamily in accordance with Mokrousov <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056577#pone.0056577-Mokrousov1" target="_blank">[38]</a>.</p

Mutations of interest characteristic for CTRI-4^XDR isolate.

1<p>Indicated in accordance with BLOSUM62 substitution matrix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056577#pone.0056577-Eddy1" target="_blank">[84]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056577#pone.0056577-Henikoff1" target="_blank">[85]</a>.</p

Comparative phylogenetic analysis of strains under study and 22 whole genomes from the NCBI database.

<p>Tree based on all SNPs of genomes. Phylogenetic tree was constructed using the Neighbor-Joining algorithm. Evolutionary distances were calculated using p-distance method. The bootstrap test was for 1000 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed.</p

Venn diagram of single nucleotide polymorphisms in three studied isolates.

<p>Venn diagram of single nucleotide polymorphisms in three studied isolates.</p