DNA operon structure optimization for the expression of fluorescent proteins in mycobacteria

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Introduction

One of the most serious challenges in the treatment of tuberculosis is the spread of strains with broad-spectrum antibiotic resistance, which necessitates the selection of medications tailored to each individual patient. A practical approach to addressing this challenge is phenotypic analysis using mycobacteriophages. It is noteworthy that as early as 1993, it was proposed to use reporter-derivative mycobacteriophages for this purpose, whose growth is indicated by an easily detectable light-emitting signal resulting from the expression of the firefly enzyme luciferase on the phage DNA [1]. Later, fluorophages — mycobacteriophages that produce fluorescent proteins — were proposed [2]. Unlike luciferase, these do not require the addition of a substrate to the mixture, do not utilize cellular ATP, and yet allow for the detection of live cells at concentrations of less than 100 per 1 mL [3]. In 2019, a methodological guide was published on determining the drug susceptibility of mycobacteria (MB) in cultures from clinical samples of patients using fluorophages [4]. However, a system for microbiological analysis similar to BACTEC can also be created based on fluorophage technology. This is important not only because of import issues under current conditions. A limitation of BACTEC is that, in this system, a fluorescent signal is generated when oxygen concentration decreases; in other words, the system reacts to the growth of any microflora. Fluorophages, on the other hand, will respond to the presence of bacterial groups on which they can grow, even if they are not closely related. The use of different bacteriophages will allow for the creation of a system that provides an optimal combination of bacterial detection and identification in clinical samples.

Furthermore, an adverse effect of cell lysis was observed even for the first luciferase-expressing mycobacteriophages. A decrease in the luminescent signal for this reason is noted for derivatives of the lytic phage TM4 [5], in contrast to analogs based on temperate phages L5 [6] and Che12 [7]. For fluorophages, cell lysis is even more undesirable. The post-translational formation (maturation) of the fluorophore via the oxidative condensation of a peptide chain consisting of three adjacent amino acids [8] takes time, ranging from tens of minutes to hours [8, 9]. Currently available fluorophages are based on derivatives of the TM4 phage with significantly reduced lytic activity [4, 5]. The fact that there has been a resurgence of interest in luminescent phages underscores the seriousness of the problem of cell lysis for fluorophages. The production of a reporter mycobacteriophage that produces the nanoluciferase NLuc and its use for analyzing the drug susceptibility of Mycobacterium tuberculosis in cultures from patient samples has been described [10]. Nluc is a convenient small enzyme (19 kDa), in contrast to the conventional firefly luciferase, which has a molecular weight of 61 kDa [11]. Like some other known luciferases [11], it does not require the presence of cellular ATP (whose synthesis can be inhibited by a drug), but it does require the presence of the external substrate furimazine. Staphylococcal bacteriophages producing the same Nluc luciferase have also been engineered and successfully used to detect Staphylococcus aureus [12]. The use of the luminophage A511:luxAB for the detection of Listeria bacteria in food products has also been reported, with a detection limit of 10 cells per 1 g of product indicated [13]. A study has also been published on the development of the reporter mycobacteriophage TM4::GeNL, which produces a conjugate of the nanoluciferase NLuc with the fluorescent protein mNeonGreen (GeNL), providing 10-fold higher sensitivity compared to NLuc [14]. GeNL combines luciferase and a fluorescent protein within a single protein molecule, enabling the resonant transfer of energy from blue-violet luminescence to a fluorophore, which emits a strong signal in the more useful green region of the spectrum. This is one of five similar NLuc-based constructs that produce light signals in blue, yellow, orange, and red [15]. It is possible that the question of the type of indicator signal remains open. On the other hand, GeNL-type hybrid luminescent-fluorescent proteins also require time for the fluorophore to mature; therefore, when produced by reporter phages, the problem of cell lysis is just as relevant as it is for fluorophages. Of particular interest is the fact that light-emitting indicator phages are already being used to detect various bacteria, not just Mycobacterium tuberculosis [1–7], a practice dating back to the 1990s. This indicates the feasibility of the aforementioned possibility of creating a phage-based analogue of BASTEC, optimally combining the detection and identification of bacteria in clinical samples. Finally, it should be noted that this article describes work on the creation of a fluorophage as an initial variant. In particular, the structures of the nanoluciferase Nluc (GenBank: YAB43984.1) and the hybrid luminescent-fluorescent protein GeNL (GenBank: BAW98162.1) are known. Genetic constructs similar to those described below can also be created for these and others.

The methodological guidelines for analyzing the drug susceptibility of Mycobacterium tuberculosis in patient culture samples [4] require obtaining fluorophage material from its developers [1, 2, 4]. Under our conditions, this clearly necessitates the development of domestically produced fluorophages, especially in light of plans to replace imports of BASTEC. For this purpose, it seems appropriate to apply the BRED (Bacteriophage Recombineering by Electroporated DNA) method — the simultaneous electroporation of purified phage DNA and synthetic DNA cassettes into Mycobacterium smegmatis cells [16, 17]. The latter must contain fluorescent protein operon (FPO) flanked on both sides by regions matching the phage DNA. This will allow the cassette to undergo recombination with the phage DNA, during which the FPO will be integrated into the phage genome. The cassette is synthesized by PCR amplification of a verified FBO from a plasmid, with addition of phage DNA segments onto its ends [16, 17]. If a lytic phage is used for this purpose, its structure must subsequently be modified using the same BRED method [16] to reduce lytic activity, which is necessary for any of the indicator signals mentioned above.

The first essential step in the process of producing a fluorophage is to design the FPO and confirm its ability to confer fluorescent properties on mycobacteria. This can be accomplished using standard genetic engineering methods within a shuttle plasmid capable of replicating in both Escherichia coli and mycobacterium — for example, the pMind plasmid [18]. The addition of a plasmid containing the FPO into M. smegmatis mycobacteria should confer fluorescence on the latter, which can be observed visually and measured using a fluorometer.

The aim of the study is to construct FPO with various possible structures within plasmids and to compare their expression in mycobacteria based on the magnitude of the fluorescent signal. This will allow us to select the FPO with the optimal structure for subsequent integration into the phage genome and the production of a fluoromycobacteriophage.

Materials and methods

In this study, E. coli XL1 Blue cells (Eurogen) were used as competent cells for transformation, genotype: recA1 endA1 gyrA96(NalR) thi-1 hsdR17 glnV44(supE44) relA1 lac [F'proAB lacI^q lacZΔM15 Tn10(Tet^R), cat. no. CC001) and M. smegmatis strain mc²155. New genetic constructs were generated using standard plasmid-based genetic engineering techniques, including DNA digestion with restriction enzymes (Sibenzyme: KpnI (cat. no. E079), FauNDI (analog of NdeI, cat. no. E009), BamHI (cat. no. E021), AhlI (analog of SpeI, cat. no. E713), and HindIII (cat. no. E073); ligation with T4 DNA ligase (Sibenzyme, cat. no. E319, or Eurogen, cat. no. LK101S), transformation of E. coli, and isolation of plasmid DNA. PCR amplification of DNA fragments was performed using the high-precision DNA polymerase Tersus (Eurogen, cat. no. PK123S) or TaqSE (Sibenzyme, cat. no. E313), with additional sequences and restriction sites introduced into the primers listed in Table 1 to ensure the production of plasmids with operons of the desired construct. All primers were synthesized at the Syntol Research and Production Facility. The structure of all operons within the plasmid was confirmed by Sanger sequencing (Eurogen) on both ends using primers targeting regions of the original pMind plasmid. For sequencing from the promoter side, the standard M13 reverse primer (5’ AGC GGA TAA CAA TTT CAC ACA GGA 3’, its 3’-end 98 bp upstream of the KpnI site at the start of the promoter-containing region), and on the terminator side, primer 9 (Table 1) — its 3’-end 38 bp downstream of the HindIII site after the terminator.

 

Table 1. Sequences of oligonucleotide primers used for FPO design

Order No.	Sequence (5’-3’)	Designation
1	ctt ggc cat atg gaa gtg att cct ccg g	Downstream primer for the promoter-containing region with an NdeI site
2	ggt gtt ggt acc ggt gac cac aac gac gcg	Upstream primer for the long variant of the promoter-containing region
3	ggc cga ggt acc ggt cga acg agg ggc atg acc	Upstream primer for the short variant of the promoter-containing region
4	gtc gta cgc aat tgt gga tcc cat tgc gaa gtg	Downstream primer for the promoter-containing region with a BamHI site
5	aaa tta cat atg aga gga tcg gga tcc g	Upstream primer for the TurboGFP protein coding region with an NdeI site
6	tcc aag act agt taa tta agc ttc att ctt ca	Downstream primer for the TurboGFP protein coding region with a SpeI site
7	cgc aat tgt gga tcc cat tgc gaa gtg att cct ttc gat cgg	Downstream primer for the promoter-containing region with RBS gp9 TM4
8	ggc ctc gga tcc gta cgc aat tgt ctt ggc cat tgc gaa gtg att	Downstream primer for the promoter-containing region for chimeric protein expression
9	caa ggc gat taa gtt ggg taa cgc cag	Reverse sequencing primer

 

The starting plasmids were pTurboGFP (Eurogen, cat. no. FP513), containing the TurboGFP fluorescent protein gene, and the shuttle plasmid pMind, provided by Brian Robertson (Addgene plasmid, cat. no. 24730; http://n2t.net/addgene:24730; RRID:Addgene_24730) [18].

Mycobacterium tuberculosis H37Rv DNA was isolated from the liquid culture pellet by thermolysis using the Express-Tub rapid mycobacterial DNA isolation kit (Syntol, cat. no. OM-518).

Electroporation of the prepared plasmids into M. smegmatis cells was performed as described in the literature [19]. Cells grown to an OD₆₀₀ of ~0.8–1.0 were washed three times with 10% glycerol in deionized water cooled in an ice bath, resuspended in the same solution at a volume of 1/20 of the original, dispensed into 200-μL aliquots, and rapidly frozen by immersion in ethanol cooled to –80°C; the cells were stored at this temperature. For electroporation, a plasmid in deionized water in an amount of 0.5–5.0 μg in a volume of no more than 10 μL was added to the cell aliquot thawed in ice. The mixture was mixed and incubated for 10 min on ice, then transferred to a 2-mm cuvette of the MicroPulser electroporator (Bio-Rad) and subjected to a 2.5-kV pulse (program Ec2). The mixture was then washed out of the cuvette with 1 mL of 7H9 medium containing OADC and 0.05% Tween-80. After 2 hours of incubation at 37°C, the mixture was added to 10 mL of the same medium containing 50 μg/mL kanamycin to selectively promote the growth of bacteria harboring plasmids. Following incubation for 3–4 days with shaking, a noticeable increase in turbidity was observed, and after centrifugation, green fluorescence could be observed when the cell pellet was illuminated with soft (365 nm) ultraviolet light.

Fluorescence measurements were performed on a CFX-96 real-time PCR instrument (Bio-Rad). For this purpose, cell suspensions, including the control (plasmid-free cells), were diluted with 7H9 medium to the same density (e.g., OD₆₀₀ ~ 0.5). Samples were dispensed in 100 μL aliquots into PCR tubes, and fluorescence intensity was measured on the CFX-96 using the following program: 3 cycles of 10 seconds at 37°C — fluorescence measurement in the FAM channel, with the lid heating turned off. Results were read using the “End point” option. The variation in fluorescence signal values for measurements of the same sample at different times on this instrument did not exceed 100 relative fluorescence units (RFU). The control cell suspension without plasmid yielded a background signal of 2500–2600 RFU; the same signal was observed for an empty well. For each sample in the series, the fluorescence value was calculated as the ratio of its signal in RFU to the control sample’s signal (signal/background).

Results

Structure of the starting plasmid and method for inserting FPO

A diagram of the pMind vector is shown in Fig. 1.

 

Fig. 1. Schematic diagram of the parental plasmid pMind.

The regions between the KpnI-BamHI and SpeI-HindIII restriction sites have been replaced with the hsp60 promoter and terminator, respectively; further, the genes encoding fluorescent proteins are to be inserted between BamHI and SpeI restriction sites.

 

This plasmid contains the mycobacterium pAL5000ori replication origin, the E. coli ColiE1ori replication origin, and the genes for resistance to kanamycin and hygromycin [18]. The objective of this study was to remove the tetR-tetO tetracycline-dependent promoter system from the pMind plasmid and replace it with the hsp60 promoter. It is strange that pMind lacks a transcription terminator, which is present in vectors used for the production of large amounts of proteins in E. coli — for example, plasmids of the pET series [20]. According to the literature, authors of studies on the expression of various proteins in mycobacteria under the hsp60 promoter also did not include a terminator in their constructs. Therefore, to avoid the synthesis of excess mRNA and a decrease in expression activity, it was decided to incorporate the hsp60 terminator, but to provide for the possibility of its removal for comparison. The unnecessary hygromycin resistance gene was removed from the pMind plasmid by digestion with HindIII and AhlI restriction enzymes (the SpeI analog in Sibenzyme). In its place, a synthetic 35-base terminator with the sites of these restriction enzymes at the ends was inserted using DNA ligase — this was the first stage in the construction of the plasmid with the FPO. Interestingly, the terminator’s function became apparent as early as during sequencing. It interrupts the polymerase reaction on which sequencing is based (Fig. 2).

 

Fig. 2. Sanger sequencing chromatogram of a pMind derivative plasmid in which the hygromycin resistance gene between the HindIII and SpeI restriction sites has been replaced with the hsp60 terminator.

The sequence of the pMind polyclon is read up to the last SpeI site (ACTAGT), the first 4 bases of the terminator (GAGG), and then the sequence is interrupted.

 

In the resulting plasmid containing the terminator, the region between the KpnI and NdeI restriction sites was then replaced with the mycobacterial hsp60 promoter, and the region encoding the TurboGFP fluorescent protein was inserted between the promoter and the SpeI site upstream of the terminator. A convenient feature of the region encoding this protein in the pTurboGFP plasmid (Eurogen, cat. no. FP513) is the presence of a HindIII restriction site downstream of this region. This allows the coding region to be amplified by PCR along with this site, with an additional SpeI site added downstream of the latter using primer 6 (Table 1). The presence of two HindIII sites on either side of the terminator will allow the latter to be deleted to assess its role in the expression of the fluorescent protein (Fig. 3). The second primer for this amplification defines the structure of the junction between the protein-coding region and the 5’-untranslated region of the hsp60 mRNA, for which no data exist in the literature with nucleotide-level precision. This makes the selection and optimization of such a structure particularly important, since the latter — like the presence or absence of a terminator — can influence the expression of the target fluorescent protein. It is known that in E. coli, protein synthesis is highly active within the pET series of plasmids [20], in many of which the ATG start codon is located within the NdeI restriction site (CATATG). On the other hand, protein expression in mycobacteria under the control of the hsp60 promoter allows for a wide range of structures in the translation start region: restriction sites, immunogenic epitopes, and signal peptides can be introduced for the secretion of proteins from cells with the corresponding modification of the N-terminus of the protein [21].

 

Fig. 3. Structure of the TurboGFP FPO within the T2 plasmid.

Shown are the amino acid sequence, restriction sites, ribosome binding site (RBS), and transcription terminator. The TurboGFP protein regions and the DNA encoding them are highlighted.

 

As a first experiment, an FPO was synthesized with a structure analogous to the pET30a plasmid (plasmid T2), where the first codon of the protein is located within the NdeI restriction site. The structure of plasmid T2 in the region encoding the TurboGFP protein is shown in Fig. 3.

Using PCR with primers 1 and 2 (Table 1), the same 374-base pair fragment containing the hsp60 promoter and the 5’-untranslated region of the mRNA was amplified from purified H37Rv MBT DNA, as described by the authors of previous studies [17, 22], and KpnI and NdeI restriction sites were introduced at its ends. It was inserted between the sites of these restriction enzymes in the aforementioned intermediate plasmid with a terminator after removing the region containing tetO-tetR from the latter (Fig. 1). Then, from the resulting plasmid, the remaining MCS linker was removed by treatment with NdeI and SpeI restriction enzymes, and the region encoding the TurboGFP protein was inserted in its place. The latter was obtained by PCR amplification of the pTurboGFP plasmid using primers 5 and 6, containing NdeI and SpeI sites, respectively. The structure of the FPO within the T2 plasmid was fully confirmed by bidirectional sequencing. Interestingly, when sequencing with primer 9, located on the remaining part of the pMind plasmid after the terminator and the second HindIII site, the entire complementary sequence of the TurboGFP protein region and the subsequent 5’-untranslated region of the mRNA are accurately read. The 35-base terminator is also read in this case and does not terminate the polymerase reaction, as occurs when it is sequenced from the other side (Fig. 2).

As it turned out, electroporation of the T2 plasmid — in which the last three nucleotides of the 5’-untranslated region of the GCA mRNA have been replaced with CAT — into M. smegmatis cells does not confer fluorescence on them. Fluorescence appears in M. smegmatis cells following electroporation with, for example, the T2L plasmid (Fig. 4), where the region before the first codon is unchanged, but a BamHI restriction site (GGATCC) is inserted after that codon, adding two small hydrophilic amino acids — glycine and serine — to the protein. This confirms the necessity to optimize the structure of the FPO.

 

Fig. 4. Structures of various TurboGFP FPO variants: with glycine and serine inserted at the N-terminus of the protein (T2S, T2L — long and short variants), T2L with the RBS of hsp60 replaced by the RBS of the gp9 gene from phage TM4 [19] (T3), and a chimeric protein with 7 amino acids from the hsp60 protein inserted into the N-terminus (T2C).

The designations are the same as in Fig. 3. The C-terminal region of the TurboGFP protein is the same in all variants as in the T2 FPO.

 

Synthesis of different variants of the TurboGFP FPO

The inclusion of an exon into the bacteriophage genome will, in any case, increase the total length of the phage DNA, which may disrupt phage particle assembly and therefore may prove risky. The insertion into the D29 phage genome of the eGFP protein coding region under the hsp60 promoter, as described in the literature [17], increased the length of the phage DNA by 671 bp. In our case, taking into account the terminator and other additional regions, it is necessary to consider the possibility of shortening the length of the FPO inserted into the phage genome.

The promoter-containing region introduced in previous studies (374 base pairs from the translation start site) [17, 22] seems to be unnecessarily long. The resulting plasmid with the T2 FPO (see above) had to be modified so that the original structure of the hsp60 operon was restored before the start of translation, and only two small amino acids — glycine and serine — were added to the N-terminus of the TurboGFP fluorescent protein (Fig. 4).

In the T2 plasmid, as in the original pTurboGFP (Eurogen, cat. no. FP513), the encoded protein contains 6 additional amino acids at the N-terminus (Fig. 3), the last two of which (the minimal glycine and serine) correspond to the BamHI restriction site in the DNA. This is very convenient because the promoter-containing region can be excised from the characterized T2 plasmid using KpnI and BamHI restriction enzymes and replaced with another region containing the specified junction structure between the non-translated and translated regions of the FPO DNA. Using purified MBT H37Rv DNA, PCR was performed with primers 2 and 4 or 3 and 4, yielding amplicons containing a promoter region of normal length and one truncated by 115 bp on the left. Their insertion into the T2 plasmid, from which the previous promoter-containing region had been removed using KpnI and BamHI restriction enzymes, yielded the T2L (standard long version of FPO) and T2S (short version of FPO) plasmids — Fig. 4, Table 2. In these, the intact hsp60 operon region — which includes the untranslated region and the first ATG codon — is followed by a BamHI site (gly and ser in the protein) and then the TurboGFP protein coding region. Other variants of the synthesized FPO constructs (Fig. 4, Table 2) were T3 (an analogue of T2L, in which the hsp60 RBS is replaced by the RBS of the gp9 gene of phage TM4 [22]) and T2C (an analogue of T2L encoding a chimeric protein in which the first methionine is followed by 6 amino acids of the hsp60 protein, then the gly-ser BamHI site, and subsequently the coding region of the TurboGFP protein). This was achieved by inserting into the T2 plasmid the amplified copies of the MBT H37Rv DNA region obtained by PCR using primers 2 and 7 (T3) or 2 and 8 (T2C). Finally, by digesting the T2L plasmid with HindIII, purifying the long fragment, and rejoining the latter with DNA ligase, a T2L analogue without a terminator — T2_t — was obtained (Fig. 3, Table 2).

 

Table 2. Characterization and fluorescent properties of the synthesized FPO variants

FPO in a plasmid	Properties	Fluorescence intensity (signal/background)
Cells without the plasmid (control)		1
T2L	BamHI site downstream of the start codon, long promoter-containing region	4,60 ± 0,10
T2S	Same, short promoter-containing region	4,82 ± 0,10
T2_t-	T2L without a terminator	2,08 ± 0,07
T3	T2L with the RBS of the gp9 gene from phage TM4	5,11 ± 0,14
T2C	T2L, chimeric protein	3,68 ± 0,11

 

The structure of all FPO within the plasmid was fully confirmed by Sanger sequencing.

Fluorescent Properties and Selection of the Optimal FPO Structure: The Case of TurboGFP

Electroporation of FPO-containing T2L, T2S, T3, T2C, and T2_t plasmids into M. smegmatis cells confers fluorescence to the cells, which can be observed visually under soft (365 nm) ultraviolet light. In this study, fluorescence was measured quantitatively using a CFX-96 real-time PCR instrument; the FAM channel proved to be the most suitable for the TurboGFP fluorophore. In general, any microplate photometer-fluorometer with a pair of excitation and emission filters suitable for the fluorophore used, or a universal monochromator, is suitable for such measurements. The results of these measurements, along with the characteristics of the obtained FPO, are presented in Table 2.

Discussion

The critical dependence of protein expression efficiency on the structure of the mRNA upstream of the translation start site proved to be quite unexpected. Replacing the three GCA nucleotides at this site in T2 with CAT blocks protein translation in mycobacteria, whereas this does not occur with the same structure in E. coli. This is an important finding revealed in this study. The results show that the promoter-containing region in T2L, identical to that described in the literature [17, 22], is in fact unnecessarily large. Its shortening in T2S does not reduce fluorescence intensity. At the same time, the total length of the FPO inserted into the phage genome turns out to be shorter than described in the literature [17]. Deletion of the terminator significantly reduces fluorescence, i.e., its presence is necessary. Replacing the T3 RBS hsp60 with the RBS of the gp9 gene from phage TM4 yields a positive effect, although not as pronounced as might be expected based on the literature [4, 23]. The T2C chimeric protein is formed but exhibits weaker fluorescence. Thus, the FPO structure in T3 can be considered optimal. It is not necessary to obtain a shortened T2S-type analog for it, since when synthesizing a recombination cassette via PCR amplification [16, 17], the position of the primers can determine any length of the region to be inserted into the phage genome.

The particular interest in obtaining a T3-structured FPO stemmed from the fact that a mCherry_bomb construct of this type had been described in the literature; it was termed a second-generation fluorophage and was characterized by a strong, stable red signal [4, 23]. In this construct, the codons of the red fluorescent protein mCherry are optimized for mycobacteria, and the RBS of the gp9 gene from phage TM4 is inserted. The exact structure of the mCherry_bomb operon’s RBS is not provided in the literature. In T3, only 7 RBS nucleotides located 10 nucleotides upstream of the ATG initiation codon have been replaced (Fig. 4 and reverse primer 7 in Table 1). However, in the TM4 phage genome (NCBI Reference Sequence: NC_003387. 1), the region preceding the gp9 protein initiation codon (cggga gaaaggagaa agccaatcAT G), highlighted in uppercase letters, differs significantly from the corresponding region for hsp60 (Fig. 3, 4) and contains a block of 16 purine nucleotides (A and G) at the RBS site. A more profound structural change upstream of the translation start site might enhance the fluorescent signal; this can be achieved.

In the T3 construct, as in the other FPO-based constructs described above, the TurboGFP-coding region can be removed by cleavage with BamHI and SpeI restriction enzymes and replaced with that of another fluorescent or indicator protein. A point of particular interest is the addition of a region for the mCherry protein, which emits red light and whose codons are optimized for mycobacteria.

Conclusion

FPO with different structures were engineered into a plasmid capable of growing in both E. coli and mycobacteria. Comparing their expression in mycobacteria based on the magnitude of the fluorescent signal allowed us to select the optimal FPO variant for subsequent integration into the phage genome — a structure in which the RBS of the hsp60 protein was replaced with that of the gp9 protein of phage TM4 (T3). The necessity of this selection was confirmed by the identification of FPO structural elements important for protein expression in mycobacteria. These include the presence of a transcription terminator and the integrity of the mRNA structure upstream of the translation start site. The latter accounts for differences, for example, in the expression of foreign proteins in E. coli and mycobacteria.

About the authors

Andrey A. Yolov

National Medical Research Center of Phthisiopulmonology and Infectious Diseases

Author for correspondence.
Email: anyol@mail.ru
ORCID iD: 0000-0003-1911-4668

Dr. Sci. (Biol.), Cand. Sci. (Chem.), leading researcher, Laboratory of immunopathology and immunodiagnostics of tuberculosis infection

Russian Federation, Moscow

Vadim V. Avdeev

National Medical Research Center of Phthisiopulmonology and Infectious Diseases

Email: vadim.avdeev@rambler.ru
ORCID iD: 0000-0002-4769-5933

researcher, Laboratory of immunopathology and immunodiagnostics of tuberculosis infection

Russian Federation, Moscow

Anastasia G. Samoilova

National Medical Research Center of Phthisiopulmonology and Infectious Diseases

Email: a.samoilova.nmrc@mail.ru
ORCID iD: 0000-0001-6596-9777

Dr. Sci. (Med.), Deputy Director

Russian Federation, Moscow

Irina A. Vasilyeva

National Medical Research Center of Phthisiopulmonology and Infectious Diseases

Email: nmrc@nmrc.ru
ORCID iD: 0000-0002-0637-7955

Dr. Sci. (Med.), Professor, Director

Russian Federation, Moscow

References

Supplementary files