Julia Bespyatykh - Academia.edu (original) (raw)

Papers by Julia Bespyatykh

Springer eBooks, 2021

Recent advances in MS/MS technology have made it possible to use proteomic data to predict protei... more Recent advances in MS/MS technology have made it possible to use proteomic data to predict protein-coding sequences. This approach is called proteogenomics, and it allows to correctly translate start and stop sites and to reveal new open reading frames. Here, we focus on using proteogenomics to improve the annotation of Mycobacteriumtuberculosis strains. We also describe detail procedures of the extraction of proteins and their further preparation for LC-MS/MS analysis and outline the main steps of data analysis.

Journal of Proteomics, Feb 1, 2019

Nowadays proteomics is one of the major instruments for editing and correcting annotation of geno... more Nowadays proteomics is one of the major instruments for editing and correcting annotation of genomic information. The correct genome annotation is necessary for omics studies of clinically relevant pathogens like Mycobacterium tuberculosis as well as for the progress in drug design and in silico biology. Here, we focused on the proteogenomic analysis of W-148 strain belonging to the Beijing B0/W148 cluster. This cluster, also known as a "successful" clone possesses unique pathogenic properties and has a unique genome organization. Taking into account high similarity of cluster strains at the genomic level we analyzed MS/MS dataset obtained for 63 clinical isolates of Beijing B0/W148. Based on H37Rv and W-148 annotations we identified 2546 proteins representing more than 60% of total proteome. A set of peptides (n = 404) specific for W-148 was found when compared with H37Rv. Start sites for 32 genes were corrected based on the combination of LC-MS/MS proteomic data with genomic six-frame translation. Additionally, we have shown the presence of peptides related to 10 genes earlier known as "pseudogenes". Significance: Mycobacterium tuberculosis is one of the most dangerous pathogens. Phylogenetically, it may be divided into major lineages and among them, lineage 2 (predominantly Beijing genotype) one of the most successful lineages with an increasing prevalence in the global population. At the same time, strains of the Beijing B0/W148 cluster, a "successful" clone of Mycobacterium tuberculosis possess even more interesting features. Only one complete genome of this cluster, W-148, present in the NCBI database (CP012090.1) and it demonstrates a number of significant differences from the well-known reference genome H37Rv. For the W-148 strain many genes are annotated as "pseudo" and no attempts were made to correct this. Thereby, in this study, we have conducted a proteomic analysis of the cluster strains and corrected current genome annotation. We hope that the data obtained will help to increase the quality of identifications in proteomic and transcriptomic analysis of M. tuberculosis Beijing B0/W148 cluster strain in subsequent studies. [6]. Today whole genome sequences for more than 200 Beijing B0/

International Journal of Molecular Sciences, Oct 30, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

mSphere, Aug 25, 2021

Reductive genome evolution is one of the most important and intriguing adaptation strategies of d... more Reductive genome evolution is one of the most important and intriguing adaptation strategies of different living organisms to their environment. Mycobacterium offers several notorious examples of either naturally reduced ( Mycobacterium leprae ) or laboratory-reduced ( Mycobacterium bovis BCG) genomes. Mycobacterium tuberculosis complex has its phylogeny unambiguously framed by large sequence polymorphisms that present unidirectional unique event changes. In the present study, we curated all known regions of difference and analyzed both Mycobacterium tuberculosis and animal-adapted MTBC species.

Journal of Clinical Microbiology, Jul 1, 2019

The Central Asia outbreak (CAO) clade is a branch of the Mycobacterium tuberculosis Beijing genot... more The Central Asia outbreak (CAO) clade is a branch of the Mycobacterium tuberculosis Beijing genotype that is associated with multidrug resistance, increased transmissibility, and epidemic spread in parts of the former Soviet Union. Furthermore, migration flows bring these strains far beyond their areas of origin. We aimed to find a specific molecular marker of the Beijing CAO clade and develop a simple and affordable method for its detection. Based on the bioinformatics analysis of the large M. tuberculosis whole-genome sequencing (WGS) data set (n ϭ 1,398), we identified an IS6110 insertion in the Rv1359-Rv1360 intergenic region as a specific molecular marker of the CAO clade. We further designed and optimized a multiplex PCR method to detect this insertion. The method was validated in silico with the recently published WGS data set from Central Asia (n ϭ 277) and experimentally with M. tuberculosis isolates from European and Asian parts of Russia, the former Soviet Union, and East Asia (n ϭ 319). The developed molecular assay may be recommended for rapid screening of retrospective collections and for prospective surveillance when comprehensive but expensive WGS is not available or practical. The assay may be especially useful in high multidrug-resistant tuberculosis (MDR-TB) burden countries of the former Soviet Union and in countries with respective immigrant communities.

Infection, Genetics and Evolution, Aug 1, 2014

ABSTRACT Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide.... more ABSTRACT Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of Mycobacterium tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of Mycobacterium tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo-biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis.

Biochemistry (Moscow). Supplement, 2018

⎯Comparative proteomic profiling of M. tuberculosis H37Rv strains cultured on two different nutri... more ⎯Comparative proteomic profiling of M. tuberculosis H37Rv strains cultured on two different nutrient media, Lowenstein-Jensen and Middlebrook 7H11, was performed using a label-free LC-MS/MS approach. It was shown that results obtained from two media possessed high convergence. The only difference was observed in the representation of fumarate reductase FrdB, its abundance was higher in the mycobacterial cells cultured on the Lowenstein-Jensen medium. The correlation analysis of biological repeats revealed the high convergence of the results obtained using the Middlebrook 7H11 medium. Thus, we can conclude that the use of the Middlebrook 7H11 medium is most appropriate in the scientific laboratory.

Scientific Reports, Mar 6, 2020

Representative functional clusters for differential genes based on transcriptomic data. Blue colo... more Representative functional clusters for differential genes based on transcriptomic data. Blue color indicates down-regulated genes. Red color indicates up-regulated genes. (A) Distribution of genes with diff expression in the main categories; (B) Detail representation of genes with diff expression in «biological process» category. " The legend for Figure 4: "Representative functional clusters for differential genes based on transcriptomic data. Blue color indicates down-regulated genes. Red color indicates up-regulated genes. (A) Distribution of genes with diff expression in the main categories; (B) Detail representation of genes with diff expression in «biological process» category. " now reads: "Multi-omics analysis of ethA gene and its product in H37Rv and RUS_B0 strains. The horizontal axis represents a schematic ethA gene (cyan) and protein (yellow), vertical axis represents transcript coverage (blue) and peptides (green). The red square indicates a frame shift mutation in the RUS-B0 strain genome. " These errors have now been corrected in the HTML and PDF versions of this Article.

Scientific Reports, Dec 17, 2019

Mycobacterium tuberculosis Beijing B0/W148 is one of the most widely distributed clusters in the ... more Mycobacterium tuberculosis Beijing B0/W148 is one of the most widely distributed clusters in the Russian Federation and in some countries of the former Soviet Union. Recent studies have improved our understanding of the reasons for the "success" of the cluster but this area remains incompletely studied. Here, we focused on the system omics analysis of the RUS_B0 strain belonging to the Beijing B0/W148 cluster. Completed genome sequence of RUS_B0 (CP030093.1) and a collection of WGS for 394 cluster strains were used to describe the main genetic features of the population. In turn, proteome and transcriptome studies allowed to confirm the genomic data and to identify a number of finds that have not previously been described. Our results demonstrated that expression of the whiB6 which contains cluster-specific polymorphism (a151c) increased almost 40 times in RUS_B0. Additionally, the level of ethA transcripts in RUS_B0 was increased by more than 7 times compared to the H37Rv. Start sites for 10 genes were corrected based on the combination of proteomic and transcriptomic data. Additionally, based on the omics approach, we identified 5 new genes. In summary, our analysis allowed us to summarize the available results and also to obtain fundamentally new data.

Pathogens, Feb 18, 2020

Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pres... more Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

Molecular Phylogenetics and Evolution, Oct 1, 2019

Scientific Reports, Jun 30, 2016

Beijing B0/W148, a "successful" clone of Mycobacterium tuberculosis, is widespread in the Russian... more Beijing B0/W148, a "successful" clone of Mycobacterium tuberculosis, is widespread in the Russian Federation and some countries of the former Soviet Union. Here, we used label-free gel-LC-MS/MS shotgun proteomics to discover features of Beijing B0/W148 strains that could explain their success. Qualitative and quantitative proteome analyses of Beijing B0/W148 strains allowed us to identify 1,868 proteins, including 266 that were differentially abundant compared with the control strain H37Rv. To predict the biological effects of the observed differences in protein abundances, we performed Gene Ontology analysis together with analysis of protein-DNA interactions using a gene regulatory network. Our results demonstrate that Beijing B0/W148 strains have increased levels of enzymes responsible for long-chain fatty acid biosynthesis, along with a coincident decrease in the abundance of proteins responsible for their degradation. Together with high levels of HsaA (Rv3570c) protein, involved in steroid degradation, these findings provide a possible explanation for the increased transmissibility of Beijing B0/W148 strains and their survival in host macrophages. Among other, we confirmed a very low level of the SseA (Rv3283) protein in Beijing B0/W148 characteristic for all «modern» Beijing strains, which could lead to increased DNA oxidative damage, accumulation of mutations, and potentially facilitate the development of drug resistance. Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB) and, according to the Global Tuberculosis Report produced by the World Health Organization (WHO), nine million people had TB in 2014 and 1.5 million died because of the disease 1. Of note, 80% of TB cases are concentrated in 22 "high-burden" countries. The Russian Federation belongs to this list and has a relatively high rate of new TB cases (80/100,000 population/year) according to WHO statistics 2. Analysis of the MTB population structure in The Russian Federation has defined three main genetic families, Ural, LAM and Beijing 3. According to earlier studies, more than 50% of all MTB strains isolated in Russia belong to the Beijing family, and a quarter of them are the Beijing B0/W148 variant 4,5. A recent systematic and critical review summarized various biological and phylogenetic features of the Beijing B0/W148 cluster 6. Strains of this cluster possess unique pathogenic properties, including stronger association with multidrug resistance and higher levels of clustering (i.e. higher transmissibility) compared with other Beijing variants, as demonstrated by a meta-analysis of studies from across the former Soviet Union 6. Additionally, members of this cluster demonstrate increased virulence in a macrophage model 7 , although in a mouse model, no increased virulence was observed 8. Beijing variant MTB strains have a unique genome organization; recently, we reported large scale chromosomal inversions spanning 350 and 550 kb segments of the chromosome 9. The presence of these inversions in Beijing B0/W148 cluster strains was confirmed by PCR, sequencing, and RFLP analysis. In addition, we identified Beijing B0/W148 cluster-specific SNPs. However, the inversions and the SNPs are insufficient to explain the success of the Beijing B0/W148 cluster. Hence, there is a particular interest in studying the proteomes of these pathogens, which will extend the genomic data by allowing detailed analyses of protein abundance, as well as protein-protein interactions.

Medicine of Extreme Situations, May 1, 2022

Tuberculosis, caused by Mycobacterium tuberculosis, remains a global burden on our country and en... more Tuberculosis, caused by Mycobacterium tuberculosis, remains a global burden on our country and entire world. According to the World Health Organization, 10 million incident cases of tuberculosis were registered in 2019. A steady increase in the drug-resistant tuberculosis aggravates the situation and appears to be the major obstacle to the fight against the disease. A thorough understanding of the pathogen physiology and virulence properties is extremely important for the development of new diagnosis methods and treatment strategies. Multiomics approaches to studying the infectious agents are indispensable in understanding the nature of the disease. Despite the availability of sufficient genomic and transcriptomic data, pathogenic potential, survival rate, persistence, immunomodulation, mechanisms underlying drug resistance and host–pathogen interaction remain poorly understood. The use of proteomic approaches has been more informative, and provides more information about the true state of the cell in various conditions. Proteomic and bioinformatic approaches helped considerably in identification and characterization of target proteins that could be used for the development of new therapeutic options. Nevertheless, OMICs data integration with simultaneous use of the system approach to studying various clinically significant mycobacterial strains makes it possible to increase knowledge about the disease mechanisms and infection control methods. The review outlines various OMICs technologies and their role in the development of the M. tuberculosis diagnostic panels.

Infekciâ i Immunitet, Jan 16, 2019

International Journal of Molecular Sciences, Sep 13, 2022

International journal of mycobacteriology, 2020

Background: Polyamines are widespread intracellular molecules able to influence antibiotic suscep... more Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Scientific Reports, Aug 23, 2017

Due to its rapid spread and association with the numerous outbreaks, the global spread of East As... more Due to its rapid spread and association with the numerous outbreaks, the global spread of East Asian lineage of Mycobacterium tuberculosis strains presents a global concern. Although there were many attempts to describe its population structure, no consensus has been reached yet. To define unbiased classification that will facilitate future studies of this lineage, we analyzed the performance and congruence of eight different genotyping schemes based on phylogenetic analysis of 1,398 strains from 32 countries using whole-genome sequencing (WGS) data. We confirm that East Asian lineage comprises two major clades, designated proto-Beijing, which harbors unusual 43-signal spoligoprofile, and Beijing, with well-known spoligoprofile (deleted signals from 1 to 34). We show that different genotyping methods give high consistency results in description of ancient Beijing strains while the classification of modern Beijing strains is significantly divergent due to star-shaped phylogeny. Using WGS data we intersect different studies and for the first time provide balanced classification with welldefined major groups and their genetic markers. Our reconstructed phylogenetic tree can also be used for further analysis of epidemiologically important clusters and their ancestors as well as white spots of unclassified strains, which are prospective areas of research.