vestimИзвестия Национальной академии наук Беларуси. Серия медицинских наукProceedings of the National Academy of Sciences of Belarus, Medical series1814-60232524-2350The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1814-6023-2023-20-1-42-57vestim-891Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEИзменчивость генов PE_PGRS и системы репарации ДНК, репликации и рекомбинаций у Mycobacterium tuberculosisVariability assessment of PE_PGRS genes and DNA repair, replication, and recombination genes in Mycobacterium tuberculosishttps://orcid.org/0000-0003-1846-6100СлизеньВ. В.SlizenV. V.

Слизень Вероника Вячеславовна – канд. мед. наук, доцент, докторант

пр. Дзержинского, 83, 220116, г. Минск

Veronika V. Slizen – Ph. D. (Med.), Associate Professor, Doctoral Candidate

83, Dzerzhinski Ave., 220116, Minsk

veronal@tut.byСурковаЛ. К.SurkovaL. K.

Суркова Лариса Константиновна – д-р мед. наук, профессор, заведующий отделом

Долгиновский тракт, 157, 220080, г. Минск

Larissa K. Surkova – D. Sc. (Med.), Professor, Head of the Department

157, Dolginovski tract, 220053, Minsk

niipulm@tut.byhttps://orcid.org/0000-0001-5683-2322ГуревичГ. Л.GurevichG. L.

Гуревич Геннадий Львович – член-корреспондент, д-р мед. наук, профессор, директор

Долгиновский тракт, 157, 220080, г. Минск

Gennady L. Gurevich – Corresponding Member, D. Sc. (Med.), Professor, Director

157, Dolginovski tract, 220053, Minsk

niipulm@tut.byБелорусский государственный медицинский университетBelarusian State Medical UniversityРеспубликанский научно-практический центр пульмонологии и фтизиатрииRepublican Scientific and Practical Center for Pulmonology and Phthisiatry2023020320232014257Copyright © Слизень В.В., Суркова Л.К., Гуревич Г.Л., 20232023Слизень В.В., Суркова Л.К., Гуревич Г.Л.Slizen V.V., Surkova L.K., Gurevich G.L.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestimed.belnauka.by/jour/article/view/891

Изучение изменчивости генов PE и PPE, а также системы репарации ДНК, репликации и рекомбинаций позволяет оценить механизмы эволюции и адаптации Mycobacterium tuberculosis.

Цель работы – изучить изменчивость генов PE_PGRS, 3R-системы (репарации ДНК, репликации и рекомбинаций) для оценки механизмов эволюционных изменений M. tuberculosis.

Проведено полногеномное секвенирование штаммов M. tuberculosis 11502 и M. tuberculosis 5005 (относились к генотипу Beijing), а также M. tuberculosis 4860 (генотип LAM), выделенных от пациентов с впервые диагностированным туберкулезом легких. Геномы загружены в международный банк геномов GеnBank, NCBI: M. tuberculosis 11502 (код доступа CP070338.1), M. tuberculosis 5005 (код доступа CP053092.1), M. tuberculosis 4860 (код доступа CP049108.1). Секвенированные геномы сравнивали со стандартным референтным штаммом M. tuberculosis H37Rv (GenBank, NCBI: NC_000962.3). В геноме M. tuberculosis 11502, относящихся к генотипу Beijing, подтипу B0/W148, кластеру 100-32, выявлены мутации в 44,4  ±  6,8 % генов (в 24 из 54), относящихся к 3R-системе, в то время как у M. tuberculosis 4860, относящихся к генотипу LAM, меньшее количество генов из системы 3R было подвержено мутациям: 29,6 ± 6,2 % (16 генов из 54). В генах 3R-системы установлен незначительный сдвиг мутаций в сторону замены на аденин и тимин, в то время как для всего генома M. tuberculosis 11502 (в сравнении с M. tuberculosis H37Rv) характерно накопление G + C. Появление мутаций в генах 3R-системы может сопровождаться субоптимальной активностью белков, ответственных за репарацию, что может приводить к увеличению частоты и спектра спонтанных мутаций. Анализ генов PE_PGRS в геноме M. tuberculosis 11502, 4860, 5005 показал, что их вариабельность варьировалась у разных представителей этого семейства генов. Было установлено высокое содержание тетрануклеотидов CGGC в большинстве генов семейства PE_PGRS, где доля CGGC варьировалась от 2,11 до 8,42 %, в то время как в среднем в геноме M. tuberculosis их доля составляла 1,62 %. Установлено, что в некоторых генах генома M. tuberculosis комбинация тетрануклеотидов CGGC вообще не встречается (Rv0011, Rv0100, Rv0460, Rv0616A, Rv0691A, Rv0722, Rv0863, Rv0909, Rv1038c, Rv1197, Rv2347c, Rv2452c, Rv3053c, Rv3320c). Конформационный анализ ДНК в местах мутаций в генах, ассоциированных с устойчивостью к противотуберкулезным лекарственным средствам, показал, что формирование вторичных структур ДНК происходит преимущественно за счет комбинаций нуклеотидов CGGC, GCGC, GGG, GGGG, CTGC, а мутации в генах возникают, как правило, в местах формирования вторичных структур ДНК (шпилек), в которых может происходить перераспределение энергии взаимодействий, зарядов, и, как следствие, могут возникать ошибки репликации и мутации. Возникновение события мутации зависит еще и от других факторов, которые могут нейтрализовать возникающие энергетические изменения в ДНК, а также влияют на точность процесса репликации, репарации (мутации в gyrA гене, в генах 3R-системы).

The variability assessment of PE/PPE genes, as well as of DNA repair, replication, and recombination system genes may drive the concept of mechanisms of Mycobacterium tuberculosis evolution and adaptation.

The aim is to study the variability of PE_PGRS genes, 3R-system genes (DNA repair, recombination, and replication) to assess the mechanisms of evolutionary changes in M. tuberculosis.

Whole genome sequencing of M. tuberculosis 11502 (the Beijing genotype subtype B0/W148 cluster 100-32), M. tuberculosis 5005 (the Beijing genotype subtype B0/W148), M. tuberculosis 4860 (the LAM genotype) strains was performed. They were isolated from patients with newly diagnosed pulmonary tuberculosis. Genomes were uploaded to the GanBank, NCBI: M. tuberculosis 11502 – access code: CP070338.1, M. tuberculosis 5005 – access code: CP053092.1, M. tuberculosis 4860 – access code: CP049108.1. A reference genome (M. tuberculosis H37Rv; NC_000962.3) was used for genetic analysis. In the M. tuberculosis 11502 genome, 44.4 ± 6.8 % of genes (24 genes out of 54) were revealed in the mutations related to the 3R system, while in M. tuberculosis 4860– 29.6 ± 6.2 % (16 genes out of 54). In the 3R system genes, a slight shift of mutations towards replacement by adenine and thymine was revealed, while the entire genome of M. tuberculosis 11502 (compared to M. tuberculosis H37Rv) demonstrated mutations, resulting in a slight accumulation of G + C. Mutations in the 3R system genes may lead to the suboptimal activity of proteins responsible for the DNA-repair, resulting in the upsurge of mutation frequency and promoting adaptive evolution. PE_PGRS genes in the genome of M. tuberculosis 11502, 4860, and 5005 exhibited a high variability and their variability diverged among different members of this gene family. A high level of tetranucleotides CGGC was found in the majority of PE_PGRS family genes, where their proportion varied from 2.11 to 8.42 %, while an average proportion of CGGC in the M. tuberculosis genome was 1.62 %. Some genes in the M. tuberculosis genome were detected to carry no tetranucleotides CGGC (Rv0011, Rv0100, Rv0460, Rv0616A, Rv0691A, Rv0722, Rv0863, Rv0909, Rv1038c, Rv1197, Rv2347c, Rv2452c, and Rv3330c). The DNA conformation analysis at the mutation sites in the genes, associated with resistance to anti-tuberculosis drugs, showed that the secondary DNA structures were mainly formed by nucleotides CGGC, GCGC, GGG, GGGG, CTGC, and mutations occurred, predominantly, at the sites of forming secondary DNA structures (hairpins) where the redistribution of energy and charges can influence the accuracy of replication and result in replication errors and a mutation event. A number of additional factors can influence the probability of a mutation event. These are the factors that can neutralize the energy changes in the DNA secondary structures, and can affect the accuracy of DNA-repair and replication (mutations in the gyrA gene, in the 3R-system genes).

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The authors declare that there are no conflicts of interest present.