Genetic analysis of the biochemical characteristics of Yersinia pestis strains of the main and minor subspecies Odinokov, Georgy Nikolaevich. Provisions for defense

Yersinia pestis main and minor subspecies

02/03/03 – microbiology

dissertations for an academic degree

Saratov – 2010 2

The work was carried out at the Russian Research Anti-Plague Institute "Microbe" Federal service on supervision in the field of consumer rights protection and human well-being"

Scientific supervisors:

Corresponding Member of the Russian Academy of Medical Sciences, Doctor of Medical Sciences, Professor Kutyrev Vladimir Viktorovich Doctor of Biological Sciences, Senior Researcher Eroshenko Galina Aleksandrovna

Official opponents:

Doctor of Biological Sciences, Professor Lidiya Vladimirovna Karpunina Doctor of Medical Sciences Natalya Ivanovna Mikshis

Leading organization: Establishment Russian Academy Medical Sciences "Research Institute of Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya RAMS"

The defense will take place “_”2010_hours at a meeting of the dissertation council D 208.078.01 for the defense of doctoral and candidate dissertations at the Federal State Institution “Russian Research Anti-Plague Institute “Microbe”” (410005, Saratov, Universitetskaya St., 46 ).

The dissertation can be found in the scientific library of the Federal State Institution “Russian Research Anti-Plague Institute “Microbe””.

Scientific secretary of the dissertation council, Doctor of Biological Sciences, senior researcher A.A. Sludsky

GENERAL DESCRIPTION OF WORK

Relevance Problems. Plague is a zoonotic natural focal especially dangerous quarantine bacterial infectious disease with the transmissible mechanism of transmission of the pathogen [Cherkassky, 1996]. Plague poses a real threat to the population due to the existence of numerous natural plague foci, some of which are located in Russian Federation and neighboring countries [Onishchenko et al., 2004]. There is a high probability of the plague pathogen being introduced into Russia from neighboring countries that are unaffected by this disease, as well as as a result of bioterrorist acts. According to WHO, more than 2,000 cases of plague are recorded annually around the world, many of which are fatal. A large outbreak of pneumonic plague in 2009 occurred in the Hainan Tibet Autonomous Region of China, which also reported a number of deaths. All these facts urgently require the development of new, highly effective methods for diagnosing the plague pathogen, based on modern technologies, and means for the prevention and treatment of the especially dangerous disease it causes.

The classifications of Yersinia pestis used so far have taken into account only morphological, cultural, biochemical and other phenotypic characteristics [Bezsonova, 1928; Borzenkov, 1938; Tumansky, 1957; Timofeeva, 1968; Kutyrev, Protsenko, 1998; Devignat, 1951] and were not without disadvantages associated with the variability of these properties. However, what has been achieved in Lately Advances in fundamental genetics and molecular microbiology make it possible to move the solution of problems of systematization of the plague pathogen to a qualitatively new level, based on the use of its molecular genetic features.

In accordance with the currently accepted domestic classification, strains of the plague pathogen are divided into the main and 4 minor (Caucasian, Altai, Gissar and Ulegey) subspecies [Timofeeva, 1985; Kutyrev, Protsenko, 1998]. According to the widespread foreign classification, Y. pestis strains, based on differences in a number of biochemical properties (the ability to ferment glycerol, reduce nitrates, and oxidize ammonia) and on a historical and geographical basis, are divided into three biovars: antiqua (antique), medievalis (medieval) and orientalis ( Oriental). According to the phenotypic characteristics, the strains of the main subspecies correspond to three biovars (ancient, medieval and oriental) accepted in the foreign classification. However, Y. pestis strains circulating in natural plague foci in the Russian Federation and neighboring countries remain unsystematized according to their belonging to specific biovars.

pestis are not capable of reducing nitrates, but ferment glycerol and arabinose.

Strains of the eastern biovar do not ferment glycerol, but actively reduce nitrates and utilize arabinose.

Strains of the main subspecies circulating in the Russian Federation and neighboring countries are, as a rule, highly virulent and have a high epidemic significance. They do not ferment rhamnose and melibiose, are not sensitive to pesticin I, and have a high level of isocitralase production. Strains of minor subspecies ferment rhamnose and melibiose, are sensitive to pesticin I, do not exhibit isocitrate-lyase activity, are selectively virulent for laboratory animals, and are of little epidemic significance.

The genetic reasons for the different expression of biochemical characteristics used in dividing Y. pestis strains into biovars and subspecies remain insufficiently studied to date. The only thing that has been reliably established is that the reason for the lack of glycerol fermentation in strains of the eastern biovar is a mutation in the glycerol-3-phosphate dehydrogenase (glpD) gene. It was shown that all strains of the eastern biovar have a deletion of 93 bp in this gene. . In the literature, there are only a few works on determining differences in the structure of the Y. pestis genes of the main and minor subspecies, encoding the reduction of nitrates and rhamnose fermentation [Kukleva et al., 2008;

2009; Anisimov et al., 2004; Zhou et al., 2004].

The reasons for the heterogeneity of plague microbe strains in terms of nutritional needs remain unknown. Strains from different natural plague foci differ in nutritional needs, determined by disturbances in intermediate metabolism genes, which can be used in a genetic scheme for differentiating Y. pestis strains from different natural plague foci.

Identification of changes in the structure of genes that underlie different expression of microbiological and biochemical traits will serve as a reliable basis for creating a genetic scheme for classifying strains of the plague pathogen, as well as for determining the main directions of intraspecific evolution of Y. pestis.

Goal of the work. Determination of the genetic basis for the different expression of biochemical characteristics used to divide strains of the plague pathogen into subspecies and biovars.

Research objectives:

To characterize the Y. pestis strains used in the work, isolated from natural plague foci in the Russian Federation and neighboring countries, according to their biochemical properties (nitrate reduction, production of isocitrate lyase, fermentation of arabinose and melibiose), which underlie the division into subspecies and biovars. To establish whether strains circulating in Russia and neighboring countries belong to certain biovars.

To study the structural and functional organization of genes encoding differential characteristics used in the division into biovars - nitrate reduction and arabinose fermentation.

To identify changes in the Y. pestis genes that determine the fermentation of melibiose and the production of isocitrate lyase, which underlie the differentiation of the main and minor subspecies of the plague pathogen.

To determine the nutritional needs of Y. pestis strains of the Caucasian subspecies and to establish the genetic basis of their auxotrophy.

To assess the prospects of using the results obtained to create a genetic scheme for the intraspecific classification of Y. pestis strains and to establish the main directions of the intraspecific evolution of this pathogen.

Scientific novelty work. Based on data from complex microbiological, biochemical and genetic analysis, it was established that the strains of the plague pathogen circulating in natural foci of the Russian Federation and neighboring countries belong to ancient and medieval biovars.

It was shown for the first time that the reason for the lack of nitrate-reducing activity in some strains of the main subspecies circulating in the Russian Federation and neighboring countries is the presence of a single nucleotide substitution G T in the position of the periplasmic nitrate reductase gene - napA, which proves that these strains belong to the medieval biovar. The lack of expression of this trait in strains of the Altai and Gissar subspecies is caused by the insertion of a thymine nucleotide (+T) at position 302 of another gene, ssuA, which leads to a shift in the reading frame and disruption of the structure of the encoded transport protein, SsuA, which is also involved in the reduction of nitrates.

It was established for the first time that the absence of arabinose fermentation in Y.

pestis of the Altai and Gissar subspecies is associated with the presence of a mutation in the regulatory gene of the arabinose operon - araC, which contains an insertion of a guanine nucleotide (+G) at position 773 from the beginning of the gene, leading to a shift in the reading frame and disruption of the structure of the regulatory protein AraC, necessary for the initiation of gene transcription arabinose operon.

For the first time, the genetic basis of the different production of isocitrate lyase in strains of the plague pathogen of the main and minor subspecies has been established, associated with the presence of an insertion of two nucleotides (+CC) in the regulatory gene iclR at position 269, which leads to the inactivation of the repressor protein of the acetate operon IclR encoded by it and is the cause of the constitutive synthesis of the enzyme isocitrate lyase in strains of the main subspecies. Strains of minor subspecies contain an intact iclR gene and are not capable of constitutive synthesis of isocitrate lyase.

It has been shown that the absence of melibiose fermentation in Y. pestis strains of the main subspecies is due to the introduction of the insertion sequence IS285 into the melB gene, which encodes the galactoside permease enzyme. In strains of minor subspecies, the IS285 insertion in the melB gene is absent.

For the first time, the genetic basis of auxotrophy of strains of the Caucasian subspecies has been identified, which is associated with the introduction of IS100 insertion sequences into the argA and aroF genes, a 10 bp insertion. into the aroG gene, insertion of a thymine nucleotide into the thiH gene and deletion of 13 bp. in the thiG gene.

The obtained molecular characteristics of Y. pestis strains of the main and minor subspecies by genes encoding the biochemical characteristics underlying the division into subspecies and biovars create the basis for the development of a genetic scheme for the intraspecific classification of the plague pathogen.

Based on the results of the work, applications for the invention “Method for determining the subspecies of plague pathogen strains by sequencing” (No. 2009116913. Priority dated May 14, 2009. A decision to issue a patent was received) and “Method for subspecies differentiation of Yersinia pestis strains using multilocus sequence typing (No. 2009146094) . Priority dated 12/11/2009).

Practical significance work. Based on the results of the work, drawn up and approved guidelines“Determination of the subspecies of plague pathogen strains based on sequencing of the rhaS and araC genes that control the fermentation of rhamnose and arabinose” (approved by the director of RosNIPCI “Microbe”. Protocol No. 6 of June 16, 2009) and “Determination of the subspecies of Yersinia pestis strains using the method of multilocus sequence typing (approved by the director of RosNIPCI “Microbe”. Protocol No. 1 of February 23, 2010).

Three strains have been deposited in the State Collection of Pathogenic Bacteria: Y. pestis KM 910 of the Altai, KM 596 of the Gissar and KM 1861 of the Ulegey subspecies as reference strains of these subspecies.

The data on the genetic organization of Y. pestis strains obtained during the study are used when delivering lectures on the subject “Genetics of the Plague Causative Agent” at specialization and advanced training courses at RosNIPCI “Microbe”.

Provisions for defense:

1. Strains of the plague pathogen of the main subspecies circulating in natural foci of the Russian Federation and neighboring countries belong to ancient and medieval biovars, as evidenced by data from a comprehensive analysis of the microbiological, biochemical and genetic properties of these strains.

2. The basis for the different manifestations of biochemical characteristics used in dividing Y. pestis strains into subspecies and biovars are different types of mutations in the genes encoding these characteristics. The lack of ability to reduce nitrates in strains of the main subspecies of the medieval biovar is associated with the presence of nonsense mutation (G T) in the napA gene of the periplasmic nitrate reductase, and in strains of the Altai and Gissar subspecies - with the insertion of a single nucleotide into the ssuA gene of the periplasmic transport protein SsuA. The absence of arabinose fermentation in strains of the Altai and Gissar subspecies is due to the insertion of a guanine nucleotide into the araC gene sequence.

The absence of melibiose fermentation by strains of the main subspecies is due to the introduction of IS285 into the melB galactoside permease gene, and the constitutive synthesis of isocitrate lyase in strains of this subspecies is due to the insertion of two nucleotides (CC) into the sequence of the iclR regulatory gene. Strains of minor subspecies contain intact melB and iclR genes.

4. The reason for the multiple nutritional needs of Y. pestis strains of the Caucasian subspecies is the inactivation of a number of genes for the biosynthesis of amino acids and vitamins. The dependence of strains of this subspecies for arginine is caused by an IS insertion into the argA gene; for phenylalanine, it is caused by a 10 bp insertion. in the aroG gene, for tyrosine - by introducing IS100 into the aroF gene, for thiamine (B1) by deletion of 13 bp. - into the thiG gene and insertion of a single nucleotide into thiH. Based on the entire complex of identified mutations for strains of the Caucasian and other subspecies, as well as biovars of the plague pathogen, characteristic genotypes were determined that can be used to create a genetic scheme for the intraspecific classification of Y. pestis.

Approbation of work. The dissertation materials were presented and discussed at the IX Interstate Scientific and Practical Conference of the CIS Member States “Modern technologies in the implementation of the global strategy for combating infectious diseases on the territory of the member states of the Commonwealth of Independent States”, Volgograd, 2008; VI International Conference “Molecular Diagnostics and Biosafety”, M., 2009; scientific and practical school conference of young scientists and specialists of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare “Modern technologies for ensuring biological safety”, May 25 – 27, 2010; at the annual final conferences of RosNIPCI “Microbe”, Saratov 2008 - 2010.

Structure and scope of the dissertation. The dissertation is presented on 157 pages of typewritten text, consists of an introduction, a literature review chapter, five chapters of own research, a conclusion and conclusions. The work is illustrated with 11 tables and 34 drawings. The bibliographic index contains 203 domestic and foreign sources.

BASIC THE CONTENT OF THE WORK

Materials and methods 111 strains of Y. pestis and Y. pseudotuberculosis were used in this work, including the main plague causative agent strain and 50 strains of minor subspecies isolated in the Russian Federation, near and far abroad. 10 strains of Y. pseudotuberculosis of various origins were also studied. The study of the cultural, morphological and biochemical properties of the strains was carried out using traditionally used methods [Practical Guide to Laboratory Diagnostics of Dangerous Infectious Diseases, 2009]. Isolation of DNA from Y. pestis strains was carried out using a standard method in accordance with the procedure given in M.U.

1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I – IV.” Analysis of the products obtained by PCR was carried out in 0.8 – 2% agarose gel in accordance with the guidelines of T. Maniatis et al. . Determination of nucleotide sequences of genes was carried out on a genetic analyzer model “CEQ 8000” (Beckman Coulter) according to the method of F. Sanger. Comparative analysis The genomes of Y. pestis and Y. pseudotuberculosis and metabolic pathways for the biosynthesis of growth factors were carried out using the BLAST algorithm of the NCBI GenBank and KEGG Metabolic Pathways databases. Phylogenetic analysis of strains was performed using Mega 4.0, PHYLIP and SplitsTree4 programs and distance matrix methods: UPGMA, FITCH, Kitch, Neighbor-Joining, Minimum Evolution.

RESEARCH RESULTS

1. Analysis of the microbiological and biochemical properties of Y. pestis strains of the main and minor subspecies from various natural plague foci. All Y. pestis strains used in the work were studied for their biochemical activity: the ability to reduce nitrates, fermentation of glycerol, arabinose, melibiose, rhamnose, production isacitrate lyase – characteristics used in dividing into subspecies and biovars. It was found that all studied strains of the main subspecies - Y. pestis ssp. pestis did not ferment rhamnose and melibiose, but produced isocitrate lyase. In contrast, all Y. pestis strains of minor subspecies fermented rhamnose and melibiose, but did not produce isocitrate lyase. The information obtained correlated with literary data on the biochemical characteristics of Y. pestis strains used to differentiate the main and minor subspecies of the plague pathogen.

When studying the biochemical characteristics used in dividing Y. pestis strains into biovars - reduction of nitrates, fermentation of arabinose and glycerol, heterogeneity in these properties was established in strains of the main and minor subspecies. Some of the strains of the main subspecies isolated in the Russian Federation and neighboring countries did not reduce nitrates, which indicated that they belonged to a medieval biovar. Strains of minor subspecies - Altai, Gissar and Ulegei also did not reduce nitrates, while Caucasian strains were positive for this trait.

According to the second differential characteristic - fermentation of arabinose, all Y. pestis strains of the main, Caucasian and Ulega subspecies studied in the work were homogeneous and utilized this monosaccharide. Strains of the Altai and Gissar subspecies did not ferment arabinose, and the genetic basis for the different expression of this trait remained unknown.

The lack of ability to ferment glycerol (another differential biochemical trait) is a characteristic feature of biovar orientalis strains. However, no foci with constant circulation of such strains have been identified in the Russian Federation and neighboring countries. There are only isolated reports in the literature about the isolation of isolated glycerin-negative strains of Y. pestis in these territories [Sagymbek et al., 2003; Onishchenko et al., 2004].

A characteristic genetic mark of biovar orientalis strains is a deletion of 93 bp. in the glpD gene – glycerol-3-phosphate dehydrogenase.

We found that all Y. pestis strains from natural plague foci in the Russian Federation and neighboring countries did not contain a 93-bp deletion characteristic of the oriental biovar in the glpD gene, which confirms the absence of orientalis biovar strains among them (Figure 1). Such strains were detected only among isolates obtained in foreign countries.

Figure 1. PCR analysis of Y. pestis strains with primers for the glpD gene: 1 – 16 – strains of the main subspecies from natural plague foci in the Russian Federation and neighboring countries; 17 – 19 – strains of the eastern biovar from foreign countries; 20 – negative control.

Arrows indicate the sizes of the formed amplifiers.

Thus, the Y. pestis strains we studied, isolated on the territory of the Russian Federation and neighboring countries, according to a complex of biochemical characteristics (glycerol fermentation, nitrate reduction) belong to ancient and medieval biovars, which correlates with the generally accepted opinion that the strains of these biovars are confined to continental foci of plague , and strains of the eastern biovar - to rat-type foci located along the ocean coasts.

During this work, much attention was paid to the study of strains of the plague pathogen of the Caucasian subspecies, since, according to a number of researchers [Bobrov, Filippov, 1997], they are the most ancient of the surviving strains of the plague pathogen and are closest to its predecessor, Y.

pseudotuberculosis. In terms of biochemical properties, strains of this subspecies turned out to be the most active among all subspecies of Y. pestis. They had nitrate-reducing activity and fermented glycerol, arabinose, rhamnose, and melibiose. Since there were conflicting data in the literature on the nutritional needs of strains of this subspecies [Martinevsky, 1969; Aparin, Golubinsky, 1989], we conducted research to clarify the nutritional needs of strains of the Caucasian subspecies.

It was established that all studied strains of the Caucasian subspecies behaved quite uniformly. They have identified a need for amino acids - phenylalanine, tyrosine, arginine and vitamin B1 (thiamine). In addition to the requirements for these growth factors, strains from the East Caucasian high-mountain plague focus also showed a dependence on leucine, while strains of the Caucasian subspecies from the Leninakan, Prisevan, Zangezur-Karabakh, and Araksin natural foci did not have such a dependence.

Since the reasons for the auxotrophy of strains of the Caucasian subspecies in amino acids and vitamin B1 (thiamine) are not known, we subsequently conducted a study of the genetic basis of the auxotrophy of strains of this subspecies.

2. Determination of the structural and functional organization of genes encoding biochemical characteristics used to differentiate biovars of the plague pathogen. The absence of the ability to reduce nitrates is a characteristic feature of Y. pestis strains of the medieval biovar, which distinguishes them from strains of ancient and eastern biovars. Strains of minor subspecies of the plague microbe circulating in natural foci in the Russian Federation and neighboring countries also differ in their denitrifying activity. However, the genetic reasons for the different manifestation of this trait in them remain unknown.

Based on a comparative computer analysis of the genomes of Y.

pestis and Y. pseudotuberculosis, presented in the NCBI GenBank database, it was found that the genes involved in nitrate reduction are combined into the nap operon, which has an identical structure and consists of six genes: napA-F and napP (Figure 2).

Other genes also involved in the reduction of nitrates were studied: narP and ssuA, encoding, respectively, the regulatory protein NarP and the transport protein SsuA Figure 2. Structure of the nap operon in Y. pestis strains As a result of a computer analysis of the nap operon genes, the presence of single nucleotide substitutions in all genes of this operon, but the most significant was the substitution of the G T nucleotide at position 613 from the beginning of the napA gene, a periplasmic nitrate reductase, which was previously identified in Y. pestis KIM and other strains of the medievalis biovar. According to computer analysis using the MEGA 4.0 program, it was established that this mutation leads to a change in the GAA TAA triplet, which is a stop codon and causes premature termination of translation of the polypeptide chain (after the 204th amino acid residue) of the molecule of this enzyme.

We identified another mutation significant for the manifestation of denitrifying activity in chromosomal gene ssuA (gene size 1123 bp), which contained an insertion of a thymine nucleotide at position 302 in microtus biovar strain 91001 (NCBI GenBank). According to computer analysis using the Mega 4.0 program, this mutation leads to a reading frame shift and disruption of the structure of the SsuA protein.

Primers were designed for the variable regions of the napA and ssuA genes, with the help of which fragments of these genes were amplified in PCR and, subsequently, their nucleotide sequence was determined. To identify the causes of different denitrifying activity in Y. pestis strains, we studied 90 Y. pestis strains of the main and minor subspecies from various natural plague foci, as well as 10 Y. pseudotuberculosis strains of various origins. As a result of the analysis, it was established that the strains of the main subspecies isolated in the Russian Federation and neighboring countries, unable to reduce nitrates, had a single nucleotide substitution G T at position 613, characteristic of biovar medievalis strains, which leads to a codon change (GAA TAA) and premature termination of translation polypeptide chain of the periplasmic nitrate reductase molecule. The presence of a characteristic nucleotide substitution at position 613, which is a genetic mark of the medievalis biovar, in some of the studied strains of the main subspecies, along with their lack of denitrifying activity, serves as a basis for classifying them as a medieval biovar.

At the same time, we did not detect the presence of this mutation in the napA gene in strains of the Altai and Gissar subspecies, which are also unable to reduce nitrates. It was found that the reason for the lack of denitrifying activity in these strains is another mutation caused by the insertion of a thymine nucleotide at position 302 of the ssuA gene of the periplasmic transport protein SsuA. This mutation was found not only in strains of the Altai and Gissar subspecies, but also in strain 91001 of microtus biovar, which is also unable to reduce nitrates.

Another important biochemical feature used for intraspecific differentiation of plague pathogen strains is arabinose fermentation. All strains of the main, Caucasian and Ulega subspecies are capable of utilizing this carbohydrate, while strains of the Altai and Gissar subspecies, as well as the microtus biovar, are not able to use it. However, the genetic reasons for the absence of manifestation of this trait in strains of the Altai and Gissar subspecies of the plague pathogen remain unidentified.

Based on a comparative computer analysis of the genomes of Y. pestis and Y.

pseudotuberculosis (NCBI GenBank) it has been established that in all strains of plague and pseudotuberculosis microbes, the arabinose operon has an identical structure and consists of six genes. It includes five structural ones - araA, araB, araH, araF, araG, and one regulatory gene araC (Figure 3).

Figure 3. Structure of the arabinose operon of Y. pestis As a result of a comparative computer analysis of the genes of the arabinose operon, as well as the araD gene in Y. pestis and Y. pseudotuberculosis strains (NCBI GenBank), it was established that there was a significant mutation in the regulatory gene araC, which contained a deletion in 112 bp (26 – 137) and a guanine insertion at position 773 in microtus biovar strain 91001, as previously found by D. Zhou et al. .

Two overlapping pairs of primers flanking the complete nucleotide sequence of this gene were designed for the variable regions of the araC gene, and its amplifications were obtained by PCR.

Sequencing of the complete sequence of the araC gene, which we carried out in natural strains of Y. pestis and 10 strains of Y. pseudotuberculosis, established that strains of only the Altai and Gissar subspecies had a mutation in the araC gene, which was associated with the insertion of a guanine nucleotide at 773 positions, while like strains of other subspecies and strains of pseudotuberculosis microbes had an intact structure of this gene. The presence of a 112 bp deletion, characteristic of microtus biovar strains, was not detected in the studied Y. pestis strains isolated in the Russian Federation and neighboring countries.

Thus, we have established for the first time that the lack of ability to utilize arabinose in strains of the Altai and Gissar subspecies is associated with the insertion of a single nucleotide in the araC gene.

3. Identification of changes in genes encoding differential characteristics used in dividing the plague causative agent into the main and minor subspecies. Strains of the plague causative agent of minor subspecies, as well as strains of Y.

pseudotuberculosis have enzymatic activity towards the disaccharide melibiose. The ability to utilize melibiosis was also found in strains of Y. pestis biovar microtus. In contrast, all highly virulent Y. pestis strains of the main subspecies do not ferment melibiosis. The genetic reasons for their lack of this biochemical activity remain unknown.

Based on a comparative computer analysis of the genomes of the Y. pestis and Y. pseudotuberculosis strains, presented in the NCBI GenBank database, we have established that the melibiose operon of the pathogens of plague and pseudotuberculosis has an identical structure and is represented by three genes – melA (YP_1469), melB (YP_1470) and melR (YP_1471) (Figure 4).

As a result of the analysis of the structure of these genes, it was established that there is a mutation important for the manifestation of the studied property in the structural gene melB, encoding the transport protein MelB – galactoside permease. In the nucleotide sequence of this gene in strains of the main subspecies - CO92, KIM, Antiqua and Nepal516 (NCBI GenBank), an insertion of an insertion sequence - IS285 after 73 nucleotides was detected. In contrast, the Y. pestis Pestoides F strain (Caucasian subspecies) and all Y. pseudotuberculosis strains have an intact melB gene structure.

A pair of primers flanking the IS285 insertion region was designed for the variable regions of the melB gene, and a fragment of this gene was amplified by PCR. In all Y. pestis strains of the main subspecies used in the work (51 strains), the amplifications obtained using this pair of primers had a size of 1648 bp, which indicated the introduction of IS285 (1324 bp) into the melB gene sequence in these strains and correlated with their lack of enzymatic activity against melibiosis. In contrast to the main subspecies, PCR revealed amplifiers of a smaller size (325 bp) in the non-main subtype strains, which indicated the intact structure of the melB gene and corresponded to their ability to ferment this disaccharide (Figure 5).

Thus, we have established that the reason for the lack of ability of Y. pestis strains of the main subspecies to ferment melibiose is a violation of the structure of the melB gene, caused by the IS285 insertion. Strains of minor subspecies contain an intact melB gene.

To differentiate strains of the main subspecies of the plague pathogen from strains of minor subspecies and pseudotuberculosis microbes, the ability of the former to constitutively synthesize the enzyme isocitrate lyase is also used.

Figure 5. PCR analysis of the melB gene in pathogen strains: 1 – 3 – Caucasian subspecies;

4 – 5 Gissar subspecies; 6 – 7 – Altai subspecies; 8 – Ulegai subspecies; 9 – 17 – main subspecies; 18 – negative control. On the left are the X174/HincII molecular weight markers.

It has been shown that strains of Y. pseudotuberculosis and Y. pestis of non-main subspecies, in contrast to strains of the main subspecies, are not capable of constitutive synthesis of this enzyme. However, the genetic reasons for differences in the expression of this trait in strains of the main and non-main subspecies are currently unknown.

According to our analysis of the genomes of the causative agent of plague and pseudotuberculosis in strains presented in the NCBI GenBank database, it was established that the structure of the acetate operon in all strains of the causative agent of plague is identical and includes three structural genes - aceA, aceB, aceK, which are under negative regulation of the gene iclR (Figure 6).

Figure 6. Structure of the acetate operon in Y. pestis strains Comparative computer analysis of the genes of the acetate operon showed the presence of a mutation in the regulatory gene iclR (843 bp) that is significant for the phenotypic manifestation of the property being studied. It was found that in Y. pestis strains CO92, KIM, Antiqua, Nepal516 (the main subspecies), the iclR gene was inactivated by the insertion of two nucleotides (+CC) at positions 269-270 from the beginning of the gene, and its size in these strains was 845 bp. The insertion (+CC) we identified in position 269-270 of the iclR gene leads to premature termination of translation of the polypeptide chain of the IclR repressor protein, which entails constitutive expression of the genes of the acetate operon and, as a consequence, a high level of isocitrate lyase synthesis.

A pair of primers was designed for the variable region of the iclR gene, with the help of which the structure of this gene was studied in 95 natural strains of Y.

pestis of the main and minor subspecies and in 10 strains of the causative agent of pseudotuberculosis. It was found that the nucleotide sequences of the iclR gene fragments in all strains of Y. pestis of the Caucasian, Altai, Gissar and Ulega subspecies are identical, amounting to 370 bp. and fully correspond to the similar sequence of pseudotuberculosis microbe strains, as well as the Pestoides F strain (NCBI GenBank), which indicates the intactness of the sequenced region of the iclR gene and correlates with the low activity of the isocitrate lyase enzyme in them. A different picture was found in strains of the main subspecies of the plague pathogen. An insertion was identified in the iclR gene - an insertion of two nucleotides (+CC) at positions 269 - 270, which leads to a shift in the reading frame of the iclR regulatory gene and, as a consequence, to the constitutive synthesis of the isocitrate lyase enzyme in Y. pestis strains of the main subspecies.

4. Establishment of the genetic basis of auxotrophy of Y. pestis strains of the Caucasian subspecies As we showed above, all strains of the Caucasian subspecies require amino acids for their growth: arginine, tyrosine, phenylalanium and vitamin B1 - thiamine. However, the genetic reasons for the auxotrophy of Y. pestis ssp.caucasica strains have not yet been studied.

At the first stage, we carried out a computer analysis of the biosynthesis pathway of arginine, tyrosine, phenylalane and vitamin B1 (thiamine) in Y. pestis and Y. pseudotuberculosis strains presented in the KEGG Metabolic Pathways database, and identified the key enzymes for the biosynthesis of these growth factors and encoding them genes. Using the BLAST algorithm, a comparative analysis of the nucleotide sequences of the genes for the biosynthesis of arginine, tyrosine, phenylalane and vitamin B1 (thiamine) was carried out in Y. pestis and Y. pseudotuberculosis strains (NCBI GenBank). According to the results of computer analysis, the mutation that we discovered in the Pestoides F strain in the structural gene of Nacetylglutamate synthase - argA, which was an insertion of the insertion sequence IS100 after the 196th nucleotide from the beginning of the gene, turned out to be significant for the manifestation of arginine dependence.

In PCR analysis using a pair of primers flanking the introduction of IS100, it was found that the size of the generated fragment of the argA gene in all strains of the plague causative agent of the main, Altai and Ulega subspecies, as well as pseudotuberculosis, was 215 bp, which corresponded to an intact gene structure. Fragments of the argA gene in Y. pestis strains of the Caucasian subspecies had a higher molecular weight(2143 bp), caused by the presence of the IS100 insertion, leading to gene inactivation.

Analysis of genes involved in the biosynthesis of aromatic amino acids showed that the aroG gene (1053 bp), encoding the DAGPS isoenzyme (3-deoxy-Darabinoheptulosonate-7-phosphate synthase), in the Pestoides F strain is inactivated by an insertion of 10 nucleotides (in positions 820 – 829), leading to a shift in the reading frame and disruption of phenylalanine synthesis.

When sequencing the aroG fragment in 95 natural strains of the main and minor subspecies, it was found that in strains of the plague microbe of the main, Altai, Gissar and Ulegey subspecies, this gene has an intact structure and encodes the functionally active protein DAGPS-. In contrast, in strains of the Caucasian subspecies of Y. pestis, a 10 bp insertion was found in the aroG gene. at positions 820–829, leading to a shift in the reading frame and disruption of the enzyme structure.

The presence of this mutation in all strains of the Caucasian subspecies correlated with their lack of ability to synthesize phenylalanine.

Another structural gene, aroF (1071 bp), encoding the DAGPSTyr isoenzyme, in the Pestoides F strain is inactivated by the introduction of the insertion sequence IS100 after 888 nucleotides from the beginning of the gene, which causes loss of the ability to synthesize tyrosine. In PCR analysis, the strains of the main, Altai, Gissar and Ulegei subspecies produced amplifiers of the aroF gene with a size of 563 bp, which corresponded to the intact structure of the gene. In strains of the Caucasian subspecies, a specific aroF gene amplification was not formed in PCR, which indicated the loss of its fragment after the introduction of the IS100 insertion sequence and correlated with tyrosine auxotrophy Structural analysis genes of the metabolic pathway of vitamin B (thiamine) biosynthesis showed the presence of significant mutations in the genes encoding the enzyme thiazole synthase - thiG and thiH, which were inactivated in the Pestoides F strain by a 13 bp deletion.

at positions 384 – 396 and insertion of a thymine nucleotide (+T) at position 552.

Our sequencing of thiG and thiH fragments in natural strains of Y. pestis revealed the intact structure of these genes in strains of the main, Altai, Gissar and Ulegey subspecies, which correlated with their ability to synthesize vitamin B1. Unlike other subspecies, strains of the Caucasian subspecies exhibited an insertion of a thymine nucleotide at position 552 of the thiH gene and a 13 bp deletion. at positions 384–396 of the thiG gene, which coincided with their lack of ability to synthesize thiamine.

Thus, we have determined for the first time the genetic basis of auxotrophy of Y. pestis strains of the Caucasian subspecies. It was established that auxotrophy for arginine is caused by the introduction of IS100 into the argA gene, for phenylalanine - by the insertion of nucleotides into aroG, for tyrosine - by the insertion of IS100 into aroF, for vitamin B1 - by a 13 bp deletion in thiG and by the insertion of a thymine nucleotide in thiH. Such mutations are not found in Y. pestis strains of other subspecies (main, Altai, Gissar and Ulegei) and therefore can be used as characteristic genetic marks of Caucasian strains.

5. Assessment of the prospects of using the obtained data to create a genetic scheme for the intraspecific classification of the plague causative agent. As a result of our research, we established the genetic basis for the different expression of biochemical characteristics used in dividing Y. pestis strains into subspecies and biovars. The variability of the napA, ssuA, araC, melB, iclR, argA, aroH, aroF, thiH and thiG genes identified during this work, along with the previously established variability of the glpD gene, as well as the variability of the rhaS regulatory gene of the rhamnose operon [Kukleva et al., 2008] can be used as the basis for a genetic scheme for classifying plague pathogen strains into biovars and subspecies. Based on a comparative analysis of the sequences of these genes, we determined the characteristic genetic features of the main (ancient, medieval, oriental biovars) and minor subspecies of Y. pestis (table). We used the variability of nucleotide sequences of genes encoding differential biochemical traits, as well as the biosynthesis of various growth factors to determine the evolutionary relationships of Y. pestis strains and construct phylogenetic trees using computer programs Mega 4.0, PHYLIP, SplitsTree 4 using distance matrix methods: UPGMA , FitchMargulis weighted mean squares, Kitch, Neighbor-Joining.

Table. Genetic characteristics of Y. pestis strains of the main and minor subspecies from natural foci of the Russian Federation and neighboring countries.

Main p/v As follows from Figure 7, the use of selected DNA targets makes it possible to fully differentiate strains of plague and pseudotuberculosis pathogens, as well as intraspecific division of Y. pestis strains not only into biovars and into main and minor subspecies, but also into separate subspecies of this pathogen.

Analysis of phylogenetic data confirms the previously stated assumption that the most ancient branch of the evolution of the plague microbe are strains of the Caucasian subspecies, which have the greatest genetic similarity to its predecessor, the pseudotuberculosis microbe (Figure 8).

Other minor subspecies – Altai, Gissar and Ulega are phylogenetically close to each other and constitute another ancient branch of the evolution of Y. pestis.

Our data indicates that the microtus strains, which Chinese researchers recently proposed to identify as a separate biovar, belong to the group of strains of the Altai and Gissar subspecies and are their variety.

Figure 7. Dendrograms (Mega 4.0 program, methods: A - UPGMA and B -Neighbor Joining) of Y. pestis strains of the main (ancient, medieval, eastern biovars) and minor (Caucasian, Altai, Gissar, Ulegey) subspecies, as well as strains of Y. pseudotuberculosis.

Figure 8. Scheme of intraspecific evolution of Y. pestis The studies carried out in this work form the basis for the development of a molecular taxonomy of the plague pathogen, the goal of which is to create a complete intraspecific taxonomy of Y. pestis based on the variability of genes for differentially significant microbiological and biochemical characteristics of the plague microbe.

CONCLUSIONS

1. Based on a comprehensive analysis of the biochemical and genetic properties of natural Y. pestis strains from various natural plague foci, it was established that the strains of the main subspecies circulating in the Russian Federation and neighboring countries belong to ancient and medieval biovars.

2. The reason for the lack of expression of a differential biochemical trait - nitrate reduction in Y. pestis strains of the main subspecies isolated in natural plague foci in Russia and neighboring countries, is the presence of a mutation - the replacement of a single nucleotide in the napA gene of periplasmic nitrate reductase, and in strains of the Altai and Gissar subspecies – insertions of a single nucleotide in the ssuA gene of the periplasmic transport protein.

3. For the first time, the genetic basis of the different expression of melibiosis fermentation traits and isocitrate lyase production in strains of the main and minor subspecies of the plague microbe has been established. It was shown that the lack of ability of strains of the main subspecies to ferment the disaccharide melibiose is due to the insertion of IS285 into the sequence of the melB structural gene, encoding galactoside permease. It has been established that the constitutive synthesis of isocitrate lyase, characteristic of the main subspecies of the plague microbe, is caused by the insertion of two nucleotides (+CC) at positions 269 – 270 of the iclR repressor gene.

4. The reason for arabino-negativity of Y. pestis strains of the Altai and Gissar subspecies is a mutation in the regulatory gene of the arabinose operon - araC, which is caused by the insertion of a single guanine nucleotide at position 773 of this gene.

5. For the first time, the genetic basis of auxotrophy of Y. pestis strains of the Caucasian subspecies has been determined. It was established that auxotrophy for arginine is caused by the introduction of IS100 into the argA gene, and for phenylalanine - by an insertion of 10 r.n. in aroG, for tyrosine – by IS100 insertion in aro F, for vitamin B1 – by a 13 bp deletion in thiG and by a thymine insertion in thiH.

6. The characteristic genotypes of the main (ancient, medieval, oriental biovars) and minor subspecies of the plague pathogen were determined, the use of which allows for intraspecific differentiation of Y. pestis. The prospects for using the results obtained to create a genetic scheme for the intraspecific classification of Y. pestis strains are shown.

LIST WORKS PUBLISHED BASED ON THE DISSERTATION MATERIALS

1. Kukleva L.M., Eroshenko G.A., Kuklev V.E., Krasnov Ya.M., Guseva N.P., Odinokov G.N., Kutyrev V.V. Comparison of the complete nucleotide sequence of the rhaS gene in strains of the plague pathogen of the main and minor subspecies // Problems of especially dangerous inf. – 2008. – Issue. 3 (97). – P. 38 – 42.

2. Odinokov G.N., Eroshenko G.A., Vidyayeva N.A., Krasnov Ya.M., Guseva N.P., Kutyrev V.V. Structural and functional analysis of nap operon genes in Yersinia pestis of different subspecies // Problems of especially dangerous inf. – 2008. – Issue. 4 (98). – P. 12 – 16.

3. Eroshenko G.A., Kukleva L.M., Vidyayeva N.A., Odinokov G.N., Shavina N.Yu., Kutyrev V.V. Genetic characteristics of minor subspecies of the plague pathogen // Modern technologies in the implementation of the global strategy for combating infectious diseases on the territory of member states of the Commonwealth of Independent States. Materials of the IX Interstate Scientific and Practical Conference of the CIS Member States. – Volgograd, 2008. – P. 71 – 73.

4. Eroshenko G.A., Vidyayeva N.A., Odinokov G.N., Kukleva L.M., Krasnov Ya.M., Guseva N.M., Kutyrev V.V. Structural and functional analysis of the araC gene in Yersinia pestis strains of various origins // Molekul. genetics, microbiol. and virusol. – 2009. – No. 3. – P. 36 – 40.

5. Kukleva L.M., Odinokov G.N., Eroshenko G.A., Kutyrev V.V. Differentiation of Yersinia pestis strains of the main and minor subspecies based on the variability of the rhaS and araC genes // Collection of materials of the VI International Conference “Molecular Diagnostics and Biosafety”. M., 2009. – P. 124.

6. Eroshenko G.A., Odinokov G.N., Kukleva L.M., Shavina N.Yu., Krasnov Ya.M., Guseva N.P., Kutyrev V.V. Variable loci of the napA, aspA, rhaS, zwf and tcaB genes as effective DNA targets for genotyping Yersinia pestis strains // Problems of especially dangerous inf. – 2010. – Issue. 2 (104). – P. 57 – 59.

7. Odinokov G.N., Eroshenko G.A. Genetic features of biochemical differentiation of Yersinia pestis strains of the main and minor subspecies // Mat.

scientific - practical school - conf. young scientists and specialists Fed. service for supervision in the field of consumer rights protection and human well-being “Modern technologies for ensuring biological safety”, May 25 – 27, 2010, pp. 289 – 291.

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Thesis

Odinokov, Georgy Nikolaevich

Academic degree:

Candidate of Biological Sciences

Place of thesis defense:

HAC specialty code:

03.02.03, 03.02.07

Speciality:

Microbiology

Number of pages:

INTRODUCTION

CHAPTER 1. LITERATURE REVIEW

1.1. Modern ideas about the organization of the genome of the 15th plague pathogen

1.2. Intraspecific classification schemes for Yersinia pestis 28 OWN RESEARCH

CHAPTER 2. MATERIALS AND METHODS

2.1. Bacterial strains used in the work and their cultivation conditions 36

2.2. Methods for studying denitrifying activity, 46 fermentation of glycerol, arabinose, rhamnose, melibiose, isocitralase production in Y. pestis strains

2.3. Determination of nutritional requirements of Y. pestis strains

2.4. Isolation of bacterial DNA

2.5. Carrying out polymerase chain reaction

2.6. Electrophoretic recording of results

2.7. Determination of nucleotide sequences of genes

2.8. Carrying out phylogenetic analysis

CHAPTER 3. ANALYSIS OF MICROBIOLOGICAL AND BIOCHEMICAL PROPERTIES OF Y. pestis STRAINS OF PRIMARY AND NON-MAIN SUBSPECTS FROM VARIOUS NATURAL

PLAGUE FOCUSES

3.1. Characteristics of Y. pestis strains from various natural plague foci according to biochemical characteristics underlying the division into subspecies and biovars

3.2. Determination of nutritional needs of K resNB strains of the Caucasian subspecies

CHAPTER 4. DETERMINATION OF STRUCTURAL-FUNCTIONAL 60 ORGANIZATION OF GENES ENCODING BIOCHEMICAL CHARACTERISTS USED FOR DIFFERENTIATION OF BIOVARS OF THE PLAGUE CAUSE

4.1. Determination of the structural and functional organization of genes of pairs 60 of the operon and other genes encoding nitrate reduction

4.2. Comparative analysis of the variability of nucleotide sequences of the genes of the arabinose operon in K. operon strains

CHAPTER 5. IDENTIFICATION OF CHANGES IN GENES ENCODING 83 DIFFERENTIAL CHARACTERISTICS USED WHEN DIVIDING THE PLAGUE CAUSE INTO MAIN AND NON-MAIN SUBSPECTS

5.1. Identification of changes in the structure of genes in strains of the causative agent of plague 83 of the main and minor subspecies encoding the fermentation of melibiosis

5.2 Determination of mutations in the isocitrate lyase biosynthesis genes in U. periasus strains

chapter 6

6.1. Comparative computer analysis of the variability of nucleotide sequences of genes involved in arginine biosynthesis

6.2. Detection of mutations in the genes for the biosynthesis of phenylalanine, tyrosine and 101 vitamin B1 (thiamine)

6.3. Study of the structural and functional state of the genes for the biosynthesis of arginine, phenylalanine, tyrosine and vitamin B1 (thiamine) in strains of the Caucasian subspecies

CHAPTER 7. ASSESSMENT OF THE PROSPECTIVENESS OF THE DATA OBTAINED FOR THE CREATION OF A GENETIC SCHEME OF INTRA-SPECIFIC CLASSIFICATION OF THE PLAGUE CAUSALUSER

Introduction of the dissertation (part of the abstract) On the topic "Genetic analysis of the biochemical characteristics of Yersinia pestis strains of the main and minor subspecies"

Relevance of the problem

Plague is a zoonotic natural focal, especially dangerous quarantine bacterial infectious disease with a transmissible mechanism of pathogen transmission [Cherkassky, 1996]. Plague poses a real threat to the population due to the existence of numerous natural plague foci, 42 of which are located in the Russian Federation and neighboring countries [Onischenko et al., 2004]. There is a danger of the plague pathogen being introduced into Russia from neighboring countries that are unaffected by this disease, as well as as a result of bioterrorist acts. According to WHO, more than 2,000 cases of plague are recorded annually around the world, many of which are fatal. A large outbreak of pneumonic plague in 2009 occurred in the Hainan Tibet Autonomous Region of China, which also reported a number of deaths. All these facts urgently require the development of new, highly effective diagnostic methods based on modern technologies for the causative agent of plague, means of prevention and treatment of the especially dangerous disease it causes.

The classifications of Yersenia pestis used so far have taken into account only morphological, cultural, biochemical and other phenotypic properties, as well as differences in the virulence of pathogen strains [Bezsonova, 1928; Berlin A.JL et al., 1938; Tumansky, 1959; Timofeeva, 1972; Kutyrev et al., 1998; Devignat, 1951; Dale et al., 2002; Cobbs et al., 2004; Dennis et al, 2004; Lazarus et al., 2004]. Such classifications, of course, contributed to the systematization of Y. pestis and generally reflect the actually existing phylogenetic relationships within this species. They have not lost their significance to this day and are still widely used in practical microbiology. However, recent advances in fundamental genetics and molecular microbiology make it possible to transfer the solution of problems of systematizing the causative agent of plague to a qualitatively new level, based on the use of its molecular genetic features. Translation of the existing classification of the plague causative agent on a genetic basis will improve the reliability, reliability and quality of systematization, as well as avoid the inherent disadvantages of classical schemes due to the variability of the phenotypic properties of the pathogen. The bacterial genome has a fairly conservative structure, protected from random changes by DNA repair systems, and therefore genetic differentiation schemes are more reliable, reproducible and have greater resolution compared to the phenotypic classifications used.

In accordance with the currently accepted domestic classification, strains of the plague pathogen are divided into the main and 4 minor (Caucasian, Altai, Gissar and Ulegei) subspecies [Kutyrev et al., 1998]. According to the widespread foreign classification, strains of 7. parabens, based on differences in a number of biochemical properties (the ability to ferment glycerol, reduce nitrates, oxidize ammonia) and on a historical and geographical principle, are divided into three biovars: anpyanaia (ancient), tesNeuainz (medieval) and openianus. (Oriental). According to the phenotypic characteristics, the strains of the main subspecies correspond to three biovars (ancient, medieval and oriental) accepted in the foreign classification. However, strains of the plague pathogen circulating in natural plague foci c. Russian Federation and neighboring countries remain unsystematized by biovar affiliation.

In terms of the expression of differentially significant biochemical characteristics, the most active are the strains of the antique biovar. They ferment glycerol and have denitrifying activity. Strains of the medieval biovar U. reese are not capable of reducing nitrates, but ferment glycerol and arabinose. Strains of the eastern biovar do not ferment glycerol, but actively reduce nitrates and utilize arabinose.

The genetic reasons for the different expression of biochemical characteristics used in dividing Y. pestis strains into biovars remain insufficiently studied to date. There is a limited number of publications on this issue in the literature. The only thing that has been reliably established is that the reason for the lack of glycerol fermentation in strains of the eastern biovar is a mutation in the glycerol-3-phosphate dehydrogenase (glpD) gene. It was shown that all strains of the eastern biovar have a deletion of 93 bp in this gene. . There is conflicting data regarding changes in genes that determine other biochemical characteristics used in the differentiation of biovars.

The division of Y. pestis into subspecies in accordance with the classification adopted in 1985 at the All-Union Meeting on the Taxonomy of the Plague Microbe is based on the unequal biochemical activity of the strains, their differences in virulence in relation to laboratory animals and different landscape-geographical locations [ Kutyrev, Protsenko, 1998; Anisimov et al., 2004]. Strains of the main subspecies are, as a rule, highly virulent and have high epidemic significance. They do not ferment rhamnose and melibiose, are not sensitive to pesticin I, and have a high level of isocitralase production. Strains of minor subspecies ferment rhamnose and melibiose, are sensitive to pesticin I, do not exhibit isocitrate-lyase activity, are selectively virulent for laboratory animals, and are of little epidemic significance.

The genetic basis of the different biochemical activity of strains causing the plague of different subspecies has been practically not studied. Abroad, this issue has been little studied due to the absence in foreign collections of strains of minor subspecies, which are distributed mainly in natural foci of the Russian Federation and neighboring countries. IN Russian literature There are only a few works on determining differences in the structure of genes, genetics of the main and minor subspecies; encoding the reduction of nitrates and fermentation of rhamnose [Eroshenko et al., 2008; Kukleva et al., 2008, 2009]. Strains of the plague pathogen of the main and minor subspecies circulating in Russia and neighboring countries remain unstudied in terms of the structure of genes involved in the reduction of nitrates, fermentation of glycerol, arabinose, melibiose, isocitrate lyase and other characteristics important for classification. The reason for the heterogeneity of plague microbe strains in terms of nutritional needs has not been established. Strains from different natural plague foci have different nutritional needs, determined by disturbances in intermediate metabolism genes, which can be used in a genetic scheme for strain differentiation ¥. rise from various natural plague foci.

Identification of changes in the structure of genes that underlie the different expression of microbiological and biochemical traits will serve as a reliable basis for creating a genetic scheme for classifying strains of the plague pathogen, as well as for determining the main directions of intraspecific evolution of U. pechnia.

Goal of the work. Determination of the genetic basis for the different expression of biochemical characteristics used to divide strains of the plague pathogen into subspecies and biovars.

Research objectives:

1. Characterize the strains of G. resiz used in the work, isolated in natural foci of plague in the Russian Federation and neighboring countries, according to their biochemical properties (reduction of nitrates, production of iso-citrate lyase, fermentation of arabinose and melibiose), which underlie the division into subspecies and biovars. To establish whether strains circulating in Russia and neighboring countries belong to certain biovars.

2. Study the structural and functional organization of genes encoding differential characteristics used in the division into biovars - nitrate reduction and arabinose fermentation.

3. To identify changes in the genes of U. ribbi, which determine the fermentation of melibiose and the production of isocitrate lyase, which underlie the differentiation of the main and minor subspecies of the plague pathogen.

4. Determine the nutritional needs of U. rhenium strains of the Caucasian subspecies and establish the genetic basis of their auxotrophy.

5. Assess the prospects of using the results obtained to create a genetic scheme for the intraspecific classification of U. paratilis strains and establish the main directions of the intraspecific evolution of this pathogen.

Scientific novelty of the work. Based on data from a comprehensive microbiological, biochemical and genetic analysis, it was established that the strains of the plague pathogen circulating in natural foci of the Russian Federation and neighboring countries belong to ancient and medieval biovars.

It was shown for the first time that the reason for the lack of nitrate-reducing activity in some strains of the main subspecies circulating in the Russian Federation and neighboring countries is the presence of a single nucleotide replacement O with T at position 613 of the periplasmic nitrate reductase gene - pair A, which proves that these strains belong to the medieval period. biovar. The lack of expression of this trait in strains of the Altai and Gissar subspecies is caused by the insertion of a thymine nucleotide (+T) at position 302 of another gene - a gene that leads to a shift in the reading frame and disruption of the structure of the encoded transport protein -8 eA, also involved in the reduction of nitrates.

For the first time, it was established that the absence of arabinose fermentation in strains of U. resia of the Altai and Gissar subspecies is associated with the presence of a mutation in the regulatory gene, the arabinose operon - agaC, which contains an insertion of a guanine nucleotide (+b) at position 773 from the beginning of the gene, leading to a shift in the reading frame and disruption of the structure of the regulatory protein AgaC, which is necessary for the initiation of transcription of the genes of the arabinose operon.

For the first time, the genetic basis of different production of iso-citrate lyase in strains of the plague pathogen of the main and minor subspecies has been established, which is associated with the presence of an insertion of two nucleotides (+CC) in the regulatory gene ShK. at position 269-270, which leads to inactivation of the acetate operon repressor protein IcIR encoded by it and is the cause of the constitutive synthesis of the isocitrate lyase enzyme in strains of the main subspecies. Strains of minor subspecies contain an intact iIII gene and are not capable of constitutive synthesis of isocitralase.

It was shown that the absence of melibiose fermentation in 7. resin strains of the main subspecies is due to the introduction of the insertion sequence 18255 in gene1B, encoding the enzyme galactoside permease. In strains of minor subspecies, insertion 1B255 in the te1B gene is absent.

For the first time, the genetic basis of auxotrophy of strains of the Caucasian subspecies has been identified, which is associated with the introduction of insertion sequences 1$>100 into the a^A and agora genes, a 10 bp insertion. into the agoO gene, by insertion of a thymine nucleotide into the d/Ru gene by deletion of 13 bp. in the gcJ gene.

The resulting molecular characterization of U. paratilis strains of the main and minor subspecies according to the genes encoding the biochemical characteristics underlying the division into subspecies and biovars creates the basis for the development of a genetic scheme for the intraspecific classification of the plague pathogen.

Based on the results of the work, applications for the invention “Method for determining the subspecies affiliation of plague pathogen strains using the sequencing method” were filed (No. 2009116913. Priority dated May 14, 2009. A positive decision was received to issue a patent) and “Method for subspecies differentiation of Yersinia pestis strains using the multilocus sequencing method - typing (No. 2009146094. Priority dated 12/11/2009).

Practical significance of the work. Based on the results of the work, methodological recommendations were drawn up and approved “Determination of the subspecies of strains of the plague pathogen based on sequencing of the rhaS and agaC genes that control the fermentation of rhamnose and arabinose” (approved by the director of RosNIPCI “Microbe”. Protocol No. 6 of June 16, 2009) and “Determination of subspecies Yersinia pestis strains using multilocus sequence typing (approved by the director of RosNIPCI “Microbe”. Protocol No. 1 of February 23, 2010).

Three strains have been deposited in the State Collection of Pathogenic Bacteria: Y. pestis KM 910 of the Altai, KM 596 of the Gissar and KM 1861 of the Ulegei subspecies as reference strains of these subspecies.

The data on the genetic organization of Y. pestis strains obtained during the study are used when delivering lectures on the subject “ Genetics of the plague pathogen"at specialization courses on especially dangerous infections at RosNIPCI "Microbe" and advanced training courses for the advanced training program for doctors in the specialty "bacteriology" at RosNIPCHI "Microbe".

Provisions for defense:

2. The basis for the different manifestations and biochemical characteristics used when dividing Y. pestis strains into subspecies and biovars are different types of mutations in the genes encoding these characteristics. The lack of ability to reduce nitrates in strains of the main subspecies of the medieval biovar is associated with the presence of a nonsense mutation (G T) in the paraA gene of the periplasmic nitrate reductase, and in strains of the Altai and Gissar subspecies - with the insertion of a single nucleotide into the ssuA gene of the periplasmic transport protein SsuA. The absence of arabinose fermentation in strains of the Altai and Gissar subspecies is due to the insertion of a guanine nucleotide into the agaC gene sequence.

3. The different biochemical activity of strains of the main and minor subspecies of the plague pathogen according to a number of differential characteristics is caused by the reduction of the genes encoding them in the main subspecies and their intactness in the minor subspecies. The absence of melibiose fermentation by strains of the main subspecies is due to the introduction of IS2S5 into the melB galactoside permease gene, and the constitutive synthesis of isocitrate lyase in strains of this subspecies is due to the insertion of two nucleotides (CC) into the sequence of the iclR regulatory gene. Strains of minor subspecies contain intact melB and iclR genes.

4. The reason for the multiple nutritional needs of Y. pestis strains of the Caucasian subspecies is the inactivation of a number of genes for the biosynthesis of amino acids and vitamins. The dependence of strains of this subspecies for arginine is caused by insertion 100 in the argA gene, and for phenylalanine - by an insertion of 10 bp. in the aroG gene, for tyrosine - by introducing IS100 into the aroF gene, for thiamine (B]) by deletion of 13 bp. - into the thiG gene and insertion of a single nucleotide into thiH. Based on the entire complex of identified mutations for strains of the Caucasian and other subspecies, as well as biovars of the plague pathogen, characteristic genotypes were determined that can be used to create a genetic scheme, intraspecific classification of Y. pestis.

Approbation of work. The dissertation materials were presented and discussed at the IX Interstate Scientific and Practical Conference of the CIS Member States “Modern technologies in the implementation of the global strategy for combating infectious diseases on the territory of the member states of the Commonwealth of Independent States”, Volgograd, 2008; VI International Conference " Molecular diagnostics and biosafety", M., 2009; scientific and practical school-conference of young scientists and specialists of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare " Modern technologies for ensuring biological safety", May 25 - 27, 2010; at the annual final conferences of RosNIPCI “Microbe”, Saratov 2008 - 2010.

Publications. 7 printed works have been published on the topic of the dissertation, 4 of them in periodicals from the “List of leading peer-reviewed scientific journals recommended by the Higher Attestation Commission of the Ministry of Education and Science of Russia.” Two patents for inventions have been issued " Method for subspecies differentiation of plague pathogen strains using sequencing method"(No. 2009116913. Priority dated 05/14/2009. A positive decision on the issuance of a patent was received) and "Method for subspecies differentiation of Yersinia pestis strains using the method of multilocus sequence typing" (No. 2009146094. Priority dated 12/11/2009).

Structure and scope of the dissertation. The dissertation is presented on 156 pages of typewritten text, consists of an introduction, a literature review chapter, five chapters of own research, a conclusion and conclusions. The work is illustrated with 11 tables and 34 drawings. The bibliographic index contains 203 domestic and foreign sources.

Conclusion of the dissertation on the topic "Microbiology", Odinokov, Georgy Nikolaevich

1. Based on a comprehensive analysis of the biochemical and genetic properties of natural Y pestis strains from various natural plague foci, it was established that the strains of the main subspecies circulating in the Russian Federation and neighboring countries belong to ancient and medieval biovars.

2. The reason for the lack of expression of the differential biochemical trait - nitrate reduction in Y. pestis strains of the main subspecies isolated in natural plague foci in Russia and neighboring countries, is the presence of a mutation - the replacement of a single nucleotide in the paraA gene of periplasmic nitrate reductase, and in Altai and Gissar subspecies - insertion of a single nucleotide in the ssuA gene of the periplasmic transport protein.

3. For the first time, the genetic basis of the different expression of melibiosis fermentation traits and isocitrate lyase production in strains of the main and minor subspecies of the plague microbe has been established. It was shown that the lack of ability of strains of the main subspecies to ferment the disaccharide melibiose is due to the insertion of IS2&5 into the sequence of the structural gene melB, encoding galactoside permease. It has been established that the constitutive synthesis of isocitrate lyase, characteristic of the main subspecies of the plague microbe, is caused by the insertion of two nucleotides (+CC) at positions 269 - 270 of the iclR repressor gene.

4. The reason for the arabino-negativity of the pestis strains of the Altai and Gissar subspecies is a mutation in the regulatory gene of the arabinose operon - agaC, which is caused by the insertion of a single guanine nucleotide at position 773 of this gene.

5. For the first time, the genetic basis of the auxotrophy of Y. pestis strains of the Caucasian subspecies has been determined. It was established that auxotrophy for arginine is due to the introduction of IS 100 into the argA gene, and for phenylalanine - an insertion of 10 r.n. in aroG, for tyrosine - by insertion of IS 100 in aro F, for vitamin Bi - by deletion of 13 bp in thiG and by insertion of thymine in thiH.

CONCLUSION

For more than hundred years of history studying the causative agent of plague after its discovery in 1894 by A. Yersen and S. Kitazato, various classification schemes for Y. pestis were repeatedly proposed, which were based on different manifestations of microbiological, biochemical and other phenotypic properties. The proposed classifications certainly corresponded to that level of knowledge and methodological approaches, which the researchers had at their disposal. So in 1928 A.A. Bezsonova, all Y. pestis strains were divided into two groups according to their ability to ferment glycerol: glycerin-negative and glycerin-positive. In 1938 A.L. Berlin and A.K. Borzenkov divided the strains of the plague pathogen into oceanic and continental races. R. Devignat in 1951, based on a number of biochemical properties (different abilities for denitrification and glycerol fermentation) and features of landscape-geographical origin, proposed the division of Y. pestis strains into three groups, which are currently designated as biovars of the plague microbe: ancient, medieval and oriental. In 1985, at the All-Union Meeting on the Taxonomy of the Plague Microbe, a unified systematic scheme of subspecies categories for the plague pathogen was adopted, based on phenotypic properties, virulence in relation to laboratory animals, and distribution area. Strains of the plague microbe circulating in the CIS countries and Mongolia were divided into five subspecies: main, Caucasian, Altai, Gissar, Ulegey (Timofeeva 1972; Kutyrev, Protsenko, 1985).

All proposed schemes were, of course, useful for researchers and contributed to the systematization of Y. pestis, and many of them (division into subspecies and biovars) are widely used at the present time. However, the rapid development of modern technologies of molecular biology, sequencing of complete genomes of pathogenic Yersinia makes it possible to solve the problem of improving the intraspecific classification of the plague pathogen at the modern molecular genetic level, based on the comparative genomics of plague pathogen strains. Translation of existing differential classification schemes for this pathogen to the genetic level will increase their resolution, efficiency and reliability, as well as improve the quality of systematization of Y. pestis species.

The genetic basis of the different biochemical activities of plague pathogen strains of different subspecies remains practically unexplored. Only individual genetic defects have been identified in the genes used for intraspecific division of Y. pestis. Thus, it has been shown that in strains of the main subspecies of the medieval biovar, the lack of the ability to reduce nitrates is associated with the presence of a mutation in the structural gene of the pairs L of periplasmic nitrate reductase, necessary for the manifestation of this property, and the inability of strains of the eastern biovar (glycerol-negative strains or oceanic race) to ferment glycerol caused by a deletion in the glycerol-3-phosphate dehydrogenase gene glpD. Evidence has been obtained that the reason for the rhamnose-negativity of all studied strains of the main subspecies is the presence of a nonsynonymous substitution of a single nucleotide in the rhaS regulatory gene of the rhamnose operon [Kukleva et al., 2008; 2009]. Regarding other genes that determine significant biochemical traits, there is either quite contradictory information or data are simply missing. Y. pestis strains of the main and minor subspecies that persist in natural foci of the Russian Federation and neighboring countries have been practically not studied for the genes of differentially significant biochemical characteristics. Studying the genetic organization of strains of various subspecies that are at different stages of the evolution of Y. pestis is an important task, since it will make it possible to determine those evolutionary transformations of the pathogen genome that led to the formation of the highly virulent bacterium Y. pestis.

To establish the genetic basis for the different expression of biochemical characteristics used in dividing strains of the plague pathogen into subspecies and biovars, we used both traditional microbiological and biochemical methods and modern methods of molecular biology - polymerase chain reaction, sequencing, and also bioinformatics methods. Internet resources were used - NGBI GenBank, KEGG Metabolic Pathways, PF AM, Modeller databases and computer programs were used: Mega 4.0 and PHYLIP with distance matrix methods.

To establish the genetic basis for the different expression of biochemical characteristics used to divide strains of the plague pathogen into subspecies and biovars, a general analysis algorithm was used. At the first stage, the expression of the microbiological or biochemical trait under study in natural strains of Y. pestis was studied. The expression of differentially significant traits (nitrate reduction, arabinose fermentation, melibiosis, isocitrate lyase production) was studied in a large number (about one hundred) Y. pestis strains from various natural plague foci.

Simultaneously, using computer analysis of strains Y. pestis KIM (medieval biovar), C092 (eastern biovar), Antiqua, Angola, Ne-pa1516 (antique biovar), 91001 (microtus biovar), Pestoides F (Caucasian subspecies) and Y. pseudotuberculosis PB1 /+, IP32953, IP31758, YPIII, the full nucleotides of whose genomes are presented in the NCBI GenBank database, we identified variable regions of genes whose products, in accordance with the KEGG Metabolic Pathways and PF AM databases, are involved in the expression of this trait. Primers were designed for variable regions of genes that were presumably the reason for the absence of phenotypic manifestation of the trait, with the help of which variable gene fragments were amplified in PCR in various natural strains of the plague causative agent. Based on a comparison of the nucleotide sequences of genes in strains that differ in the expression of the studied trait, mutations were identified that caused the absence of this trait.

Using this algorithm, we studied the structural and functional organization of genes, the products of which are involved in the manifestation of differential characteristics that underlie the division of Y. pestis strains into biovars - nitrate reduction and arabinose fermentation. A comparative computer analysis of gene pairs was performed operon, as well as the pagP and ssuA genes - regulatory (NarP) and transport (SsuA) proteins involved in the reduction of nitrates, in strains presented in the NCBI GenBank database, as well as in a large number (about a hundred) natural strains of Y. pestis and minor subspecies isolated in various natural plague foci in the Russian Federation, near and far abroad.

It has been established that the reason for the lack of denitrifying ability in some strains of the main subspecies is the presence of a single nucleotide substitution G to T at position 613 of the gene pair A, encoding the protein - periplasmic nitrate reductase. The reason for the inability to reduce nitrates by strains of the Altai and Gissar subspecies is different and is associated with the presence of a mutation in the transport protein gene - ssuA, which in these strains at position 302 contains a single nucleotide insertion (+T). The data obtained, along with the biochemical characteristics of the strains (the ability to reduce nitrates, fermentation of arabinose and glycerol), allowed us to conclude that strains of ancient and medieval biovars circulate in the Russian Federation and neighboring countries, while strains of the eastern biovar are detected only among strains from foreign countries.

Apparently, the reason for the lack of nitrate-reducing activity in microtus biovar strains is the same mutation as in strains of the Altai and Gissar subspecies - the insertion of a single nucleotide in the ssu gene. We have established that a mutation in the pairA gene - the insertion of a single pair A gene at position 1021 cannot be the reason for the lack of expression of the trait in microtus strains [as suggested by D. Zhou et al., 2004], since we also identified it in strains of the Caucasian subspecies , as well as in pseudotuberculosis strains 7, which reduce nitrates. "

For the first time, a structural and functional analysis of the genes of the arabinose operon was carried out in a large number of natural strains of the plague causative agent, and the complete nucleotide sequence of the regulatory gene agaC, involved in the regulation of the expression of arabinose fermentation, was determined. It has been established that the reason for the absence of this trait in the Altai and Gissar subspecies is the presence of a single nucleotide insertion in the agaC gene at position 773 from the beginning of the gene. Strains of the main, Caucasian and Ulegay subspecies do not contain such a mutation, which correlates with their ability to ferment arabinose.

A study was carried out of the structure of genes encoding the fermentation of melibiose and the production of isocitrate lyase, which are used in dividing Y. pestis strains into the main and minor subspecies. Previously, the genetic determination of the different manifestations of these properties in strains of the main and non-main subspecies has not been studied. There are no data on this issue in the literature.

A comparative computer analysis of the nucleotide sequence of melibiose fermentation genes (melA, melB and melR) in Y. pestis and 7. pseudotuberculosis strains presented in the NCBI GenBank database showed the presence in the melB gene (1232 bp), which determines the synthesis of galactoside permease, insertion of the IS2&5 insertion sequence (1322 bp) after 73 bp. in plague pathogen4 strains C092, KIM, Antiqua, Nepal516. In other strains of 7 pestis Angola, 91001, Pestoides F, and in all strains of 7 pseudotuberculosis, an intact melB gene structure was found. In PCR analysis using calculated primers flanking the IS2SJ insertion region, specific fragments of the melB gene were obtained from a large number of natural strains of the plague pathogen. In the studied 7 pestis strains of non-main subspecies - Caucasian, Altai, Gissar and Ulegei - PCR produced amplifications of 325 bp in size, which corresponded to the intact structure of the melB gene. In contrast, in strains of the main subspecies the fragments had a larger size of 1648 bp, which indicated the introduction of an insertion sequence into this gene and correlated with their lack of enzymatic activity against melibiose. Thus, for the first time we have established a genetic cause; different expression of a differential feature - fermentation of melibiose in strains of the plague pathogen of the main and minor subspecies, which is associated with a violation of the structure of the melB gene in strains of the main subspecies due to the introduction of the insertion sequence IS2&5.

To differentiate Y. pestis strains of the main subspecies from non-main ones and the causative agent of pseudotuberculosis, their ability to constitutively synthesize the enzyme isocitrate lyase is used. Comparative computer analysis of the nucleotide sequences of the genes of the acetate operon (aceA, aceB, aceK, iclR) in G. pestis and Y. pseudotuberculosis strains presented in the NCBI GenBank database showed the presence of an insertion of two nucleotides in iclR (gene size 843 bp) (+CC) at position 269-270 from the beginning of the gene in Y. pestis strains C092, KIM, Antiqua, Nepal516. In contrast, other strains of the plague microbe Angola, 91001, Pestoides F and all strains of pseudotuberculosis have an intact structure of the iclR gene. Sequencing of the variable region of the iclR gene, carried out by us in natural strains of Y. pestis, revealed the presence of the same mutation in the strains of the main subspecies of Y. pestis - the insertion of two nucleotides (+CC) at positions 269 - 270, in contrast to the minor subspecies in which an intact the structure of this gene. The identified mutation leads to gene inactivation and disruption of the structure (and function) of the repressor protein of the acetate operon IclR, associated with the loss of the C-terminal end of the domain (148 - 271 aa) that binds the inducer molecule. This leads to constitutive expression of the genes of the acetate operon and correlates with high activity of the isocitrate lyase enzyme in strains of the main subspecies of Y. pestis.

Previously, it was suggested that the most ancient strains of the plague microbe are strains of the Caucasian subspecies [Bobrov, Fillipov, 1997; Kukleva et al., 2002]. Their study is of significant interest, since it allows us to determine those stages evolutionary changes genome, which led to the transformation of a saprophytic enteropathogenic bacterium - a pseudotuberculosis microbe - into a highly virulent systemic pathogen with a fundamentally different mechanism of pathogen transmission. Our results indicate that the Caucasian subspecies is the most active in terms of differential biochemical characteristics and contains intact genes (paraA, ssuA, glpD, araC, melB, iclR), like the causative agent of pseudotuberculosis. This confirms the greater proximity of the Caucasian subspecies to its predecessor, Y. pseudotuberculosis, compared to other subspecies of Y. pestis.

We also carried out microbiological studies to study the nutritional needs of Y. pestis strains of the Caucasian subspecies, since there was conflicting information on this issue in the literature. It was established that all studied strains of the Caucasian subspecies behaved uniformly. They have identified a need for two aromatic amino acids - phenylalanine and tyrosine, as well as the amino acid arginine and vitamin Bi (thiamine). In addition to the requirements for arginine, phenylalanine, tyrosine, and vitamin B1, strains from the East Caucasian high-mountain plague focus also showed a dependence on leucine, while strains of the Caucasian subspecies from the Leninakan, Prisevan, Zangezur-Karabakh, and Araksin natural foci of such dependence did not have.

To establish the genetic basis of the auxotrophy of strains of the Caucasian subspecies, using the KEGG and PFAM databases, an analysis of metabolic pathways and identification of enzymatic systems involved in the biosynthesis of aromatic amino acids - phenylalanine, tyrosine, the amino acid arginine and vitamin Bt (thiamine) were carried out. Significant mutations were found in the biosynthesis genes of arginine (argA), phenylalanine (aroG), tyrosine (aroF), and vitamin Bl (thiH, thiG) in strains of the Caucasian subspecies. In the structural gene a^A, an insertion of the insertion sequence \S100 of barely 196 bp was detected, which is the reason for the auxotrophy of the Caucasian subspecies for arginine. In the ago gene

Thus, we have established for the first time the genetic basis of auxotrophy of strains of the Caucasian subspecies. The obtained genetic characteristics of strains of the Caucasian subspecies for the genes of differential biochemical characteristics ((ggarA, vvi, glpD, agaC, me1B, yuSh) and for the genes of biosynthesis of growth factors (agA, agoC, agor, ¿/g/C and ShN) indicate the greatest antiquity of this subspecies, as well as long period its evolution, independent of other subspecies of U. re, M "ya.

Based on a detailed molecular genetic analysis of natural strains of U. resib from various plague foci, the genetic characteristics of strains of other subspecies circulating in Russia, near and far abroad were also determined. The use of the identified genetic mutations in the genes paraA, agaC, glpD, mA, and SHR makes it possible with high efficiency and reliability to determine the belonging of U. pebis strains to the main or minor subspecies, and the strains of the main subspecies to one of three biovars - ancient, medieval or oriental. Characteristic genotypes of each of these taxonomic units—the species Y. pestis—were identified.

The variability of the genes of the pairs A, ssu, araC, melB, iclR, argA, aroH, aog F, thiH and thiG, established during this work, along with the previously identified variability of the glpD gene, was used to reconstruct the phylogenetic scheme of the evolution of the main and minor plague pathogen strains subspecies, which confirmed the antiquity of the Caucasian strains, as well as other minor subspecies of Y. pestis (Figure 34).

As follows from this diagram, the causative agent of plague originates from the pseudotuberculosis microbe and is a branch of the evolution of this enteropathogenic Yersinia. The most ancient subspecies of Y. pestis is phylogenetically closer to Y. pseudotuberculosis compared to other subspecies of the plague microbe. Another ancient branch of the evolution of Y pestis is represented by the Ulega, Altai and Gissar subspecies, which are genetically close to each other and, apparently, separated from the general trunk of evolution into a single group, which later split into separate subspecies. Apparently, the most ancient among them is the Ulegai subspecies, which contains smaller number mutations in life-support genes (in particular, it lacks a mutation in the agaC gene) compared to strains of the Altai and Gissar subspecies. The latter are phylogenetically close to each other, as well as to strains of the microtus group, which contain the same mutations in the genes of differential biochemical characteristics as the strains of the Altai and Gissar subspecies. We were the first to suggest that microtus strains belong to the group of Altai-Gissar subspecies.

С092 (orientalis)

KIM 776 (meclievalis) (meclievalis)

NepalSlö (antiqua) subsp. ulegeica subsp. hismrica

91001 (microtus) subsp. altaica.

231, 680 subsp. caucasica

Y. pseu dotu bereu losis

Figure 34. Scheme of intraspecific evolution of U. rheaT^ya

The studies carried out in this work form the basis for the development of a molecular taxonomy of the plague pathogen, the goal of which is to create a complete intraspecific taxonomy of Y. pestis based on the variability of genes for differentially significant microbiological and biochemical characteristics of the plague microbe.

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Introduction to the work

Relevance of the problem. Plague is a zoonotic natural focal, especially dangerous quarantine bacterial infectious disease with a transmissible mechanism of pathogen transmission [Cherkassky, 1996]. Plague poses a real threat to the population due to the existence of numerous natural plague foci, some of which are located in the Russian Federation and neighboring countries [Onshtsenko et al., 2004]. There is a high probability of the plague pathogen being introduced into Russia from neighboring countries that are unaffected by this disease, as well as as a result of bioterrorist acts. According to WHO, more than 2,000 cases of plague are recorded annually around the world, many of which are fatal. A large outbreak of pneumonic plague in 2009 occurred in the Hainan Tibet Autonomous Region of China, which also reported a number of deaths. All these facts urgently require the development of new, highly effective methods for diagnosing the plague pathogen, based on modern technologies, and means for the prevention and treatment of the especially dangerous disease it causes.

Classifications used so far Yersinia pestis only morphological, cultural, biochemical and other phenotypic characteristics were taken into account [Bezsonova, 1928; Borzenkov, 1938; Tumansky, 1957; Timofeeva, 1968; Kutyrev, Protsenko, 1998; Devignat, 1951] and were not without disadvantages associated with the variability of these properties. However, recent advances in fundamental genetics and molecular microbiology make it possible to move the solution of problems of systematization of the plague causative agent to a qualitatively new level, based on the use of its molecular genetic features.

In accordance with the currently accepted domestic classification, strains of the plague pathogen are divided into the main and 4 minor (Caucasian, Altai, Gissar and Ulegey) subspecies [Timofeeva, 1985; Kutyrev, Protsenko, 1998]. According to the common foreign classification, strains Y. pestis Based on differences in a number of biochemical properties (the ability to ferment glycerol, reduce nitrates, oxidize ammonia) and on a historical and geographical basis, they are divided into three biovars: antiqua (antique), medievalis (medieval) and orientalis (eastern). According to the phenotypic characteristics, the strains of the main subspecies correspond to three biovars (ancient, medieval and oriental) accepted in the foreign classification. However, strains Y. pes-

4 tis circulating in natural foci of plague in the Russian Federation and neighboring countries remain not systematized according to their belonging to certain biovars.

According to the expression of differentially significant biochemical characteristics, the most active are the strains of the ancient biovar. They ferment glycerol and have denitrifying activity. Strains of the medieval biovar ". pestis are not able to reduce nitrates, but ferment glycerol and arabinose; Strains of the eastern biovar do not ferment glycerol, but actively reduce nitrates and utilize arabinose.

Strains of the main subspecies circulating in the Russian Federation and neighboring countries are, as a rule, highly virulent and have a high epidemic significance. They do not ferment rhamnose and melibiose, are not sensitive to PESTICE I, and have a high level of isocitralase production. Strains of minor subspecies ferment rhamnose and melibiose, are sensitive to pesticin I, do not exhibit isocitrate-lyase activity, are selectively virulent for laboratory animals, and have little epidemic significance.

Genetic reasons for different expression of biochemical characteristics and strains used in dividing Y. pestis into biovars and subspecies remain insufficiently studied to date. The only thing that has been reliably established is that the reason for the lack of glycerol fermentation in strains of the eastern biovar is a mutation in the glycerol-3-phosphate dehydrogenase gene (glpD). It was shown that in this gene all strains of the eastern biovar have a deletion of 93 bp. . In the literature there are only a few works on determining differences in gene structure Y. pestis main and minor subspecies, responsible for the reduction of nitrates and fermentation of rhamnose [Kukleva et al., 200 2009; Anisimov et al., 2004; Zhou et al., 2004].

The reasons for the heterogeneity of plague strains in terms of nutritional needs remain unknown. Strains from various natural plague foci differ in nutritional needs, determined by disturbances in intermediary metabolism genes, which can be used in a genetic scheme for diferentiation of strains Y. pestis from various natural plague foci.

Identification of changes in the structure of genes that underlie different expression of microbiological and biochemical traits will serve as reliable information for creating a genetic scheme for classifying strains of the plague pathogen, as well as for determining the main directions of intraspecific evolution Y. pestis.

5 Purpose of the work. Determination of the genetic basis for the different expression of biochemical characteristics used to divide strains of the plague pathogen into subspecies and biovars.

Research objectives:

Characterize the strains used in the work Y. pestis, isolated in natural foci of plague in the Russian Federation and neighboring countries, according to biochemical properties (reduction of nitrates, production of isocitrate lyase, fermentation of arabinose and melibiose), which underlie the division into subspecies and biovars. To establish whether strains circulating in Russia and neighboring countries belong to certain biovars.

To study the structural and functional organization of genes encoding differential characteristics used in the division into biovars - nitrate reduction and arabinose fermentation.

Identify changes in genes Y. pestis, determining the fermentation of melibiosis and the production of isocitrate lyase, which underlie the differentiation of the main and minor subspecies of the plague pathogen.

Determine the nutritional needs of strains Y. pestis Caucasian subspecies and establish the genetic basis of their auxotrophy.

Assess the prospects of using the results obtained to create a genetic scheme for intraspecific classification of strains Y. pestis and establishing the main directions of intraspecific evolution of this pathogen.

Scientific novelty of the work. Based on data from complex microbiological, biochemical and genetic analysis, it was established that the strains of the plague pathogen circulating in natural foci of the Russian Federation and neighboring countries belong to ancient and medieval biovars.

It was shown for the first time that the reason for the lack of nitrate-reducing activity in some strains of the main subspecies circulating in the Russian Federation and neighboring countries is the presence of a single nucleotide substitution G -»T at position 613 of the periplasmic nitrate reductase gene - pair, which proves that these strains belong to a medieval biovar. The lack of expression of this trait in strains of the Altai and Gissar subspecies is caused by the insertion of a thymus nucleotide (+T) at position 302 of another gene - ssuA, which leads to a shift in the reading frame and disruption of the structure of the encoded transport protein - SsuA, which is also involved in the reduction of nitrates.

It was established for the first time that the absence of arabinose fermentation in strains] pestis Altai and Hissar subspecies is associated with the presence of a mutation in the regulatory gene of the arabinose operon - yeahS, which contains an insertion of a guanine nucleotide (+G) at position 773 from the beginning of the gene, leading to a frameshift and disruption of the structure of the regulatory protein AgaC, which is necessary to initiate transcription of the genes of the arabinose operon.

For the first time, the genetic basis of different production of isocitr-lyase in strains of the plague pathogen of the main and minor subspecies has been established, associated with the presence of an insertion of two nucleotides (+CC) in the regulatory gene iclR in position 26! 270, which leads to the inactivation of the acetate operon repressor protein IclR encoded by it and is the cause of the constitutive synthesis of the enzyme isocitr lyase in strains of the main subspecies. Strains of minor subspecies contain a shtact gene iclR and are not capable of constitutive synthesis of isocitrate lyase.

It has been shown that the absence of melibiose fermentation in strains Y. pestis the main subtype is due to the introduction of an insertion sequence IS285 gene melB, encoding the enzyme galactoside permease. In strains of non-major subspecies, the IS2&5 insertion in the gene melB absent.

For the first time, the genetic basis of auxotrophy of strains of the Caucasian subspecies, which are associated With introduction of insertion sequences IS100 genes argA And aroF, insert 10 bp. into the gene aroG, insertion of thymine nucleotide gene thiHv. 13 bp deletion in the gene thiG.

Obtained molecular characteristics of strains Y. pestis the main and neo-new subspecies based on genes encoding the biochemical characteristics that underlie the division into subspecies and biovars, creates the basis for the development of a genetic scheme for the intraspecific classification of the plague pathogen.

Based on the results of the work, applications for the invention “Method for determining the subspecies of plague pathogen strains by sequencing method” were filed (No. 2009116913. Priority dated May 14, 2009. A decision was received to issue a patent and “Method for subspecies differentiation of strains Yersinia pestis by multilayer sequence typing method (No. 2009146094. Priority from 11.12.2009).

Practical significance of the work. Based on the results of the work, methodological recommendations “Determining the subspecies of plague pathogen strains based on gene sequencing” were drawn up and approved rhaS And yeahS, controlling the fermentation of rhamnose and arabinose" (approved by the director of RosNIHR "Microbe". Protocol No. 6 of June 16, 2009) and "Determination of subspecies;

7 strain characteristics Yersinia pestis by multilocus sequence typing method (approved by the director of RosNIPCI “Microbe”. Protocol No. 1 of February 23, 2010).

Three strains are deposited in the State Collection of Pathogenic Bacteria: Y. pestis KM 910 of the Altai, KM 596 of the Gissar and KM 1861 of the Ulega subspecies as reference strains of these subspecies.

Data on the genetic organization of strains obtained during the study Y. pestis are used when delivering lectures on the subject “Genetics of the Plague Causative Agent” at specialization and advanced training courses at RosNIPCI “Microbe”.

Provisions for defense:

Strains of the plague causative agent of the main subspecies, circulating in natural foci of the Russian Federation and neighboring countries, belong to ancient and medieval biovars, as evidenced by data from a comprehensive analysis of the microbiological, biochemical and genetic properties of these strains.

The basis for the different manifestations of biochemical characteristics used when dividing strains Y. pestis subspecies and biovars contain different types of mutations in the genes encoding these traits. The lack of ability to reduce nitrates in strains of the main subspecies of the medieval biovar is associated with the presence of a nonsense mutation (G - T) in the gene pair periplasmic nitrate reductase, and in strains of the Altai and Gissar subspecies - with the insertion of a single nucleotide into the gene ssuA periplasmic transport protein SsuA. The absence of arabinose fermentation in strains of the Altai and Gissar subspecies is due to the insertion of a guanine nucleotide into the gene sequence yeahS.

The different biochemical activity of strains of the main and minor subspecies of the plague pathogen according to a number of differential characteristics is caused by the reduction of the genes encoding them in the main subspecies and their intactness in the minor subspecies. The absence of melibiose fermentation by strains of the main subspecies is due to the introduction into the gene melB galactoside permease, and the constitutive synthesis of isocitrate lyase in strains of this subspecies is due to the insertion of two nucleotides (CC) into the sequence of the regulatory gene iclR. Strains of minor subspecies contain intact genes melB And iclR.

Cause of multiple nutritional requirements of strains Y. pestis Caucasian subspecies is the inactivation of a number of genes for the biosynthesis of amino acids and vitamins. The dependence of strains of this subspecies for arginine is caused by the insertion of IS70O into the gene argA, for phenylalanine - insert 10 bp. into the gene aroG, for tyrosine - insertion IS100 into the gene aroF, for thiamine (BO by deletion of 13 bp - in the gene thiG and insertion of a single

8th nucleotide in MN. Based on the entire complex of identified mutations for strains of the Caucasian and other subspecies, as well as biovars of the plague pathogen, characteristic genotypes were determined that can be used to create a genetic scheme for intraspecific classification Y. pestis.

Approbation of work. The dissertation materials were presented and discussed at the IX Interstate Scientific and Practical Conference of the CIS Member States “Modern technologies in the implementation of the global strategy for combating infectious diseases on the territory of the member states of the Commonwealth of Independent States”, Volgograd, 2008; VI International Conference “Molecular Diagnostics and Biosafety”, M., 2009; scientific and practical school of the conference of young scientists and specialists of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare “Modern technologies for ensuring biological safety”, May 25 - 27, 2010; at the annual final conferences of RosNIPCI “Microbe”, Saratov 2008 - 2010.