Genetic polymorphism of the р66 gene of Borrelia bavariensis circulating in a natural focus and its association with Ixodid tick-borne borrelioses in the population of the Middle Urals

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Introduction

Ixodid tick-borne borrelioses (ITBB) are a group of chronic or recurrent naturally endemic vector-borne infections that affect various human systems and organs [1]. The causative agents of these diseases are spirochetes of the Borrelia burgdorferi sensu lato complex, which includes more than 20 species of Borrelia [2]. Three of them (B. garinii, B. afzelii, and B. bavariensis) are the most common pathogens of ITBB in Eurasia. Based on long-term incidence rates, ITBB ranks first among naturally endemic infections [3–5].

Borrelia species of the B. burgdorferi s. l. complex form complex three-component parasitic systems that are inextricably linked in Eurasia to the life cycles of the taiga (Ixodes persulcatus ) and European forest (I. ricinus) ticks [1]. Their existence is impossible without feeders, which serve as reservoir hosts for Borrelia . The primary hosts for the pre-imaginal developmental stages of these ticks are small forest mammals [1, 6].

In Russia, one of the most widespread pathogens of tick-borne diseases is B. bavariensis. This relatively recently described distinct biological species of Borrelia [7] was previously known as the widespread Eurasian genetic subgroup NT29 of B. garinii [1]. B. bavariensis has been detected in situ in Ixodes ticks and their reservoir hosts of several species [8–14].

The pathogenicity factors of Borrelia bacteria primarily include their surface membrane proteins [15]. One of these is the P66 protein, which is encoded by the chromosomal gene . This protein mediates the pathogen’s adhesion to the endothelial cells of mammalian blood vessels [16] and binds to the β1- and β3-chains of integrins [15], which facilitates the penetration and dissemination of Borrelia within the mammalian host [17]. P66 protein binds to the anti-phagocytic receptor SIRPα on the surface of macrophages, which facilitates Borrelia ’s evasion of the mammalian immune response [18].

The variability of B. bavariensis p66 gene has been virtually unstudied, and among its alleles there may be variants that cause ITBB in humans more frequently than others. The aim of this article is to present the main results of a series of studies on the polymorphism of p66 gene in B. bavariensis isolates circulating in the natural focus, the findings of the main sections of which have been described in detail previously [19–21]. The objective is to analyze the variability of p66 gene in B. bavariensis isolates from components of the pathogen’s parasitic system (vectors and reservoir hosts) in comparison with the frequency of nucleotide sequence variants of this gene in isolates from patients with ITBB.

Materials and methods

The nucleotide sequences of p66 gene region (280 bp), which encodes the immunodominant hypervariable surface loop near the C-terminus of P66 protein, were studied in 238 B. bavariensis isolates stored in the Borrelia museum of the Laboratory of Infection Vectors, based at the State Collection of Microorganisms—Pathogens of Human Infectious Diseases of Pathogenicity Groups II–IV from SCM—Gamaleya. They were obtained in 1992–2006 and include 27 isolates from patients at the Perm Regional Infectious Diseases Hospital (58°00′ N, 56°15′ E) and 211 isolates, of which 123 were from two species of ixodid ticks (I. persulcatus, I. trianguliceps), and 88 were from eight species of small mammals (Myodes glareolus, Alexandromys oeconomus, M. rutilus, Sylvaemus uralensis, Craseomys rufocanus, Agricola agrestis, Sicista betulina, Sorex araneus) from a natural focus located in the Middle Urals (58°32′ N, 57°43′ E).

The isolation and labeling of isolates, their cultivation on BSK medium, primary identification and re-identification, polymerase chain reaction (PCR), and purification of the resulting PCR products have been described previously [19–21]. Amplicons of p66 gene region were sequenced using the ABI PRISM Big Dye Terminator v. 3.1 reagent kit (Applied Biosystems) and subsequent analysis of the reaction products on the ABI 3500xL DNA Analyser (Applied Biosystems) at the Genome Shared-Use Center (V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences). The results were analyzed using the BLAST service and the Chromas, UGENE, MEGA11, and Jalview software packages. Nucleotide and amino acid alignments were performed using the Muscle method. Dendrograms were constructed using the Jones–Taylor–Thornton model by the maximum likelihood method with a bootstrap value of 1,000 repetitions.

Statistical analysis of the results was performed using the χ² test in GraphPad Prism 8. Differences were considered significant at p < 0.05. Fifty-two nucleotide sequences of the p66 gene region were deposited in the NCBI GenBank database (accession numbers OP561855–OP561858; OR183688–OR183707; OR620166–OR620180; PP940149–PP940161).

Results

Based on the updated scheme of the main transmission routes of Borrelia in natural foci (Fig. 1), the diversity of genetic structures in p66 gene region was investigated in 238 isolates of the Eurasian subgroup B. bavariensis from vectors — Ixodes ticks and reservoir hosts of Borrelia — small mammals that sustain the parasitic system of this pathogen in the Middle Urals, as well as from patients with ITBB. On two extensive working dendrograms, the nucleotide sequences of p66 gene region, as well as the amino acid sequences of P66 protein encoded by this gene, were grouped into 12 major clusters. To present and visualize the results of the study, we built a dendrogram of the amino acid sequences of P66 protein fragment, encoded by the corresponding 12 variants of p66 gene nucleotide sequences—i.e., alleles of the Eurasian subgroup B. bavariensis, which are numbered in descending order of the number of similar isolates (Fig. 2).

 

Fig. 1. Transmission routes of the most common Eurasian pathogens of ITBB (B burgdorferi s. l.) within their parasitic systems, as well as human infections. Solid arrows indicate primary transmission routes of Borrelia; dotted arrows indicate secondary and possible rare routes. Components of the Borrelia parasitic system: 1 — engorged female ticks; 2 — oviposition; 3 — unfed larvae; 4 — small mammals; 5 — unfed nymphs; 6 — medium-sized mammals and birds; 7 — adult unfed ticks (imago); 8 — large mammals.

 

Fig. 2. A dendrogram of 47 amino acid sequences of P66 protein fragments from isolates selected for study from each group of source species. Isolates from humans with ITBB are marked with blue circles, from background reservoir host species — with red diamonds, from small mammals of rare host species — with purple squares, and from adults and nymphs of I. persulcatus — by pink and red triangles, and from nymphs of I. trianguliceps — by yellow squares. The serial numbers of allelic variants are indicated in the squares.

 

The spectrum of allelic variants and their proportions: among infected vectors and reservoir hosts of Borrelia, the profile was found to be virtually identical (Fig. 2). Alleles No. 1 and No. 2 predominated, as was the case among isolates from human patients. The greatest diversity of allelic variants was found in isolates obtained from small mammals of background species, which can be explained by the greater representativeness of the number of isolates studied from Borrelia sources in this group (Table). The number of isolates of each of the remaining 10 variants accounts for 0.4–7.6% of their total number in the group. Based on the absolute values of the samples we compared, no statistically significant differences in the frequency of these variants were detected among B. bavariensis isolates from Ixodes ticks and small mammals (χ² = 0.0002–0.344; p = 0.558–0.988). However, they are clearly evident (χ² = 8.644; p = 0.003) between Borrelia isolates from patients with ITBB and from adult I. persulcatus ticks — the sole source of human infection in the Perm Krai.

 

The proportion of variants 1 and 2 of P66 protein in the studied B. bavariensis isolates from patients with ITBB and the main groups of the pathogen sources within pathogen’s parasitic system

Source of B. bavariensis isolates	Designation in Fig. 2.	Number of isolates studied	Of these, the number of isolates containing P66 protein fragment variants No. 1 and No. 2		Source
			abs.	%
People with ITBB	Hs	27	26	96,3	[19]
Unfed adult taiga ticks	Ip	58	39	67,2	[21]
Taiga tick nymphs	Ipn	40	28	70,0	[21]
Rodents of the most abundant species—reservoir hosts of Borrelia	Cg and Mo	73	51	69,9	[20]
I. trianguliceps tick nymphs	Itn	25	19	76,0	[21]

 

The presence of various amino acid sequence variants of P66 protein in isolates carrying B. bavariensis alleles identified by us and other researchers is reflected in the dendrogram (Fig. 3). It includes data on the structure of this protein region in isolates and reference strains selected by us from the NCBI GenBank database based on their geographic location and having non-synonymous differences in nucleotide sequence of the corresponding p66 gene region. Most of these deposited sequences belonged to alleles No. 1 and No. 2. A few isolates were found with 3 different structural variants of this gene (and, accordingly, of P66 protein amino acids), which differ from the 12 allelic variants we described in B. bavariensis isolates; as well as in strains of the European subgroup of Borrelia of this species (according to NCBI GenBank data). The generalized dendrogram of all detected variants of P66 protein amino acid sequences generally indicates that it differs in isolates of the European genetic subgroup of B. bavariensis (Fig. 3, univariant cluster A) from the Eurasian subgroup (more polyvariant cluster B).

 

Fig. 3. A dendrogram of the amino acid sequences of P66 protein in B. bavariensis isolates, as well as isolates from the European and Eurasian subgroups of Borrelia of this species, selected from the NCBI GenBank database based on their isolation in different countries and regions and on differences in the nucleotide sequence of p66 gene region. Isolates from humans with ITBB are marked with blue circles, from background reservoir host species — with purple diamonds, from imagoes and nymphs of I. persulcatus — with pink and red triangles, and from nymphs of I. trianguliceps—with yellow squares. The serial numbers of allelic variants are indicated in the squares. Cluster A represents the European subgroup of B. bavariensis; cluster B represents the Eurasian subgroup. The names of the strains are given in parentheses, and GenBank accession numbers for the p66 nucleotide sequences are given in square brackets; the geographical location of isolation is indicated for each strain. Yellow stars indicate single amino acid structures of P66 B. bavariensis that are likely allelic variants of this pathogen.

 

Amino acid sequences of P66 protein, identical to 2 of the 12 allelic variants of Eurasian subgroup B. bavariensis that we identified in the Middle Urals, have been found in the NCBI GenBank database in isolates from patients with ITBB in Japan, as well as in I. persulcatus and Dermacentor sp. from various regions of Russia (Arkhangelsk and Tomsk regions, Perm Krai) and other countries (China, Mongolia; Japan) (Fig. 3). The international database also contains several other p66 gene sequences, which may lead to certain amino acid substitutions in the structure of the corresponding protein. Most of these occurred in the 482–522 amino acid region of P66 protein’s surface loop (Fig. 4).

 

Fig. 4. Amino acid sequence alignment of the P66 protein for identified allelic variants of B. bavariensis (variant numbers are listed before the isolate designation) and GenBank isolates representing single P66 structures. Conservation — quantitative assessment of conservation of the physicochemical properties of amino acids (AMAS method). Quality — alignment quality (BLOSUM62 matrix). Consensus — the consensus sequence of P66 protein is indicated. The extracellular loop of P66 protein is indicated.

 

Proteins from certain isolates with nucleotide sequences closely resembling allelic variant No. 1 differ in the presence, at positions 497, 508, and 513, of the following amino acids (respectively): polar serine, polar negatively charged glutamic acid, and nonpolar isoleucine, as well as from the reference strain PBi B. bavariensis by several amino acid substitutions (p.461N>I only in isolate Hs-10 ; p.469I>L; p.492A>T; p.494N>S; p.499T>A; p.514I>T). The sequence of this variant was identical to that of the Japanese isolate J14, obtained from a patient with ITBB (Fig. 4). Proteins from isolates similar to allelic variant No. 2 have different amino acids at positions 497, 508, and 513 (respectively): nonpolar alanine and glycine, and polar threonine. They differ from the protein structure of strain PBi in a manner analogous to the first variant. An identical sequence of P66 protein fragment of this allele was detected in the Hiratsuka isolate obtained from a patient with ITBB in Japan. Isolates with the 10 remaining identified allelic variants have single amino acid substitutions (as well as a threonine insertion in isolate Sb-3174 after position 490) in their proteins (Fig. 4). Analysis of data in the NCBI GenBank database showed that p66 gene fragment encoding P66 protein in the Japanese isolate J20T (obtained from a patient with ITBB) is completely identical to allelic variant No. 7 (Fig. 4).

As noted above, the NCBI GenBank database contains three unique sequences that differ from the 12 B. bavariensis allelic variants we identified in terms of both nucleotide sequence and amino acid composition. These are represented by isolates J15, Mng4702, and JAASAAF1014 (Figs. 3, 4). Isolate J15, obtained in Japan from an infected human, bears a strong resemblance to allelic variant No. 2 in terms of these structures, with the exception of an amino acid substitution at position 483: polar glutamine is replaced by polar, positively charged lysine. In the Japanese strain JAASAAF1014, isolated from I. persulcatus tick, the amino acid structure of P66 protein, in addition to the substitution at position 497 characteristic of allelic variant No. 1 (see above), reveals polar threonine at position 499 instead of nonpolar alanine. The composition of this protein fragment in the Mongolian isolate Mng4702 (from taiga ticks) differs from all the listed variants by a substitution at position 473 of polar threonine with nonpolar alanine (Fig. 4). The described substitutions in the structure of P66 protein in the 3 original isolates allow them to be considered potential additional allelic variants of the Eurasian subgroup of B. bavariensis, especially since they differ at various positions by a number of substitutions (p.469I>L; p.492A>T; p.494N>S; p.497T>A; p.514I>T) from the reference European strain PBi.

Overall, the results of the studies conducted indicate that certain allelic variants of p66 gene identified by us are widespread among isolates of the Eurasian subgroup B. bavariensis, as well as the possible presence of alleles with a unique structure of this gene in the pathogen’s parasitic systems found in various geographical settings.

Discussion

In natural foci, the circulation of the most epidemiologically significant pathogens of ITBB in the B. burgdorferi s. l. group is inextricably linked to the complex, long- term life cycles of the tick vectors, which involve changes in their hosts — the reservoir hosts of Borrelia. Figure 1 illustrates the main, secondary, and possible rare routes of transmission of ITBB pathogens, which is based on general quantitative patterns of the Borrelia population at different stages of their parasitic systems and elaborates on the general scheme of ITBB pathogen circulation by Yu.S. Balashov and E.I. Korenberg [1, 6].

In the Middle Ural forests of the Perm Krai, humans are attacked by unfed adults and, in rare cases, nymphs of I. persulcatus (I. ricinus, like other species of Ixodes ticks capable of infecting humans with ITBB pathogens, is absent in this region) [1, 21]. Unfed adults acquire Borrelia only through transstadial transmission from engorged, infected, metamorphosed nymphs, to which the pathogen is transmitted in the same way from larvae, as well as through parasitism on infected reservoir hosts (Fig. 1, positions 3, 4, and 6) [22]. Transovarial transmission of Borrelia from fed, infected females to unfed larvae is unlikely (Fig. 1, positions 1–3) [23, 24]. Therefore, such larvae don’t transmit Borrelia to their hosts but become infected themselves while feeding on them: primarily on small mammals (Fig. 1, position 4) [1, 22]. At the same time, unfed larvae (and nymphs) can acquire various allelic variants of Borrelia , which determines the likelihood of their subsequent transstadial transmission all the way to the final stage of the tick’s full life cycle — that is, unfed adults that bite humans.

Infected Ixodes ticks and small mammals of various species — hosts of B. bavariensis, as well as some other pathogenic B. burgdorferi sensu lato [25–27] — have virtually identical sets of allelic variants of p66 gene region, i.e., fragments of P66 protein (Fig. 2). This suggests that in the parasitic system of ITBB pathogen, there is no selective adaptation of specific Borrelia allelic variants under the influence of the internal environment of their vectors and hosts. At least half of the B. bavariensis isolates we studied from each of the main source groups in the natural focus had allelic variant No. 1 or No. 2. Such alleles were also present in the majority of infected adult unfed taiga ticks capable of attacking humans (table), which explains the frequency of human infection in the Middle Urals with precisely these variants of the ITBB pathogen.

The nymphs of taiga tick, which have a wide range of hosts [1, 22, 26] and the greatest diversity of transmission routes for Borrelia within the parasitic system of B. bavariensis (and presumably for other common pathogens of ITBB in the B. burgdorferi s. l. group; Fig. 1), play a key role in the transmission of various alleles of the Borrelia p66 gene circulating in the natural focus. At the same time, Borrelia can persist in the bodies of reservoir hosts for virtually their entire lives [26, 28]. Consequently, overwintered animals (along with metamorphosed pre-imago ticks) that became infected during the preceding spring-summer stage (or stages) of the epizootic process resume transmission after seasonal dormancy, which is due to the autumn-winter period of tick inactivity [1, 20, 26]. These factors contribute to the maintenance of relatively stable polymorphism and allelic ratios of p66 gene of the ITBB pathogen, including the predominance in a specific parasitic system of variants that infect humans most frequently (table).

This doesn’t mean that the allelic variants of p66 gene found in the B. bavariensis parasitic system — which have been detected only in small mammals (Nos. 10–12) or in adult I. persulcatus ticks (Nos. 4–9), but not in isolates from human patients—cannot cause ITBB in humans. Such cases could likely be identified if rare variants of P66 protein structure were found with significantly higher frequency in ticks that bite humans, or (and) if a large number of isolates from patients with ITBB were examined. For example, allelic variant No. 7 was identified in the J20T isolate of B. bavariensis obtained from a patient with ITBB in Japan. Isolate J15, obtained in the same country from an infected human, presumably represents another allelic variant of this pathogen. In regions where the primary vector of Borrelia is the I. ricinus tick and B. bavariensis of the European genetic subgroup predominates [9], its parasitic system may include the circulation of P66 protein fragment variants that differ from those found in Eurasia (Fig. 3).

Overall, the presented results of our studies of a large group of Borrelia isolates from their main sources in the parasitic system of B. bavariensis in the Middle Urals, combined with an analysis of data from the international NCBI GenBank database, confirm the broad geographic distribution of the Eurasian genetic subgroup of this ITBB pathogen (including p66 alleles Nos. 1 and 2). Genetically, it is more polymorphic than its European subgroup [29, 30]. The circulation of Borrelia of the Eurasian subgroup B. bavariensis, which is predominantly associated with I. persulcatus [5, 8, 21], may involve other species of Ixodidae ticks (e.g., I. pavlovskyi [26]). For example, the I. trianguliceps tick, which also inhabits this region and does not bite humans, was found to have a similar set of allelic variants to B. bavariensis, with alleles 1 and 2 being predominant (table). This indicates the additional role of I. trianguliceps ticks, which parasitize small mammals of the same species as the pre-imaginal stages of I. persulcatus, in maintaining the genetic variability of the B. bavariensis ITBB pathogen in the natural focus, where taiga ticks play a key role [21].

In the P66 protein of allelic variants No. 1 and 2, the amino acid substitutions S497A, E508G, and I513T were detected. These substitutions likely affect the conformation of its surface loop, which, as a result, contributes to the enhancement of this protein’s function, including the initial interaction (adhesion) of Borrelia with recipient cells. The biochemical mechanism of this process requires further study. Nevertheless, the two identified structures of P66 protein, which determine the onset of infectious process caused by B. bavariensis, can be considered as candidate targets for the development of a prophylactic drug to prevent ITBB in humans. In this regard, for the development of such a polyvalent drug, it is important to study the degree of similarity of functionally analogous protein structures in other ITBB pathogens most prevalent in Eurasia: B. garinii, B. afzelii, and B. miyamotoi.

Conclusion

In the Middle Urals, at least 12 allelic variants of p66 gene region—which encodes the eponymous protein of the pathogen’s bacterial cell, essential for the initiation of infectious process of B. bavariensis — circulate among the components of B. bavariensis parasitic system (tick vectors and mammalian reservoir hosts). At all stages of the epizootic process, the frequency of Borrelia isolates with different variants of p66 gene sequences remains unchanged. The majority of them have two variants (Nos. 1 and 2) of nucleotide sequences of this gene region, which have been detected in almost all isolates of this ITBB pathogen from patients infected in the Perm Krai. Such alleles of p66 gene of B. bavariensis are also widespread in a number of other regions of Eurasia.

The nucleotide sequences of alleles No. 1 and 2, which encode their amino acid sequences, differ significantly from the other identified allelic variants. They lead to amino acid substitutions that may affect the conformation of the surface loop of P66 protein, which presumably enhances its adhesive function. This allows these variants of P66 protein structure to be considered as potential targets for the development of prophylactic drugs to prevent the development of ITBB in humans bitten by ticks.

About the authors

Kristina A. Golidonova

National Research Center for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya

Author for correspondence.
Email: kristi.dekor@mail.ru
ORCID iD: 0000-0003-4832-6248

researcher; Laboratory of vectors of infections

Russian Federation, Moscow

Edward I. Korenberg

National Research Center for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya

Email: edkorenberg@yandex.ru
ORCID iD: 0000-0002-4452-4231

Dr. Sci. (Biol.), Professor, principal researcher; Laboratory of vectors of infections

Russian Federation, Moscow

References

Supplementary files