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.     

Synthesis and biological properties of α-thymidine 5′-aryl phosphonates

Diethyl(N-arylaminocarbonyl)methyl phosphonates have been obtained by the reaction of diethylphosphonoacetic acid imidazolides with methyl-4-aminobenzoate or 3,5-bis(trifluoromethyl)phenylamine. Their treatment with Me₃SiBr in DMF led to a mixture of the corresponding (N-arylaminocarbonylmethyl)phosphonic acids and their monoethyl esters. After separation, they were condensed with 3′-O-acetyl-α-thymidine, which, after the removal of the acetyl protecting group, gave (α-D-thymidine-5′-yl)-[4-aminocarbonyl-, methoxycarbonyl-, or carboxy)phenylaminocarbonyl]methyl phosphonates and (α-D-thymidine-5′-yl)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It was shown that the compounds are stable under different conditions, low toxic (in Vero and K-562 cell cultures), and capable of penetrating into K-562 cells. Only ethyl (α-D-thymidine-5′-yl)-[4-(methoxycarbonyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the growth of the laboratory strain M. tuberculosis H37Rv.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Microbial diversity of Late Pleistocene Siberian permafrost samples

A metagenomic study of the Kolyma lowland permafrost samples, 20–35 thousand years, performed using a Geneclean for Ancient DNA kit (Bio101, United States), revealed 8 phylotypes which belonged to the phyla Actinobacteria and Proteobacteria. Analysis of the 16S rRNA gene clone library showed that most of the clones (48% and 29%) were represented by the genera Arthrobacter and Bradyrhizobium, respectively. For the first time microorganisms of the genera Williamsia, Bradyrhizobium, Filomicrobium and Hansschlegelia were observed in the ancient microbial communities of these ecosystems. Analysis of the isolates 16S rRNA genes revealed the presence of the microorganisms—the representatives of the phyla Firmicutes and Actinobacteria phylogenetically related to known species and being obvious representatives of novel taxa. In situ electron-microscope analysis of total preparations of the studied samples showed the presence of intact bacterial cells of different morphotypes.     

Spoligotyping of mycobacterium tuberculosis isolates obtained from patients in the Samara region

The spoligotyping of 138 M. tuberculosis isolates obtained on the territory of the Samara region made it possible to classify them as belonging to 27 groups. The largest cluster including 94 strains (68.1%) belonged to Peking spoligotype. As shown with the use of the commercial system INNO-LiPA, 78 out of 134 strains (58.2%) had mutations in different sites of gene pro B. The analysis of the markers of resistance to rifampicin in Peking spoligotype revealed that 65 (83.9%) out of 90 such cultures had mutations in gene pro B, i.e. potentially these strains had multiple antimicrobial resistance. This work demonstrated the prevalence of Peking spoligotype in a prison antituberculosis colony (81.6%).     

Typing of Mycobacterium tuberculosis cultures from Samara region by the variable number of tandem repeats

To type Mycobacterium tuberculosis (MT) strains by the variable number of tandem repeats (VNTR), 136 MT cultures, isolated in 5 main general therapeutic laboratories and penitentiary antituberculosis institutions of the Samara region, were studied. Gene typing revealed that the greatest number of MT strains (73) belonged to VNTR type 42435. It was followed by VNTR type 22232, found in 13 isolated cultures. Among the MT cultures of the most widely spread VNTR type 42435, rifampicin-resistant strains prevailed (57 strains, i.e. 78%), while strains belonging to VNTR type 22232 were mainly sensitive to rifampicin (84%). VNTR typing of MT typing may be useful for epidemiological studies in the field of phthisiology.     

Detection of the Bartonella DNA by the method of nested PCR in patients after tick bites in Novosibirsk region

Arthropod-borne bacterial pathogen Bartonella DNA was detected in human blood cells after tick bites in summer 2003 and 2004 in Novosibirsk region by nested PCR with primers specific to groEL gene of HSP60 protein. Comparative assay of 190 p.b. of long PCR fragment revealed that the nucleotide sequences might belong to Bartonella henselae and Bartonella quintana.