Application of the GW module for the immobilization of the red fluorescent protein RFP on peptidoglycan from lactic acid bacteria  Lactococcus lactis  and  Lactobacillus acidophilus

Olga Yu. Dobrynina; Добрынина Ольга Юрьевна; Abdul-Khamit  M. Umyarov; Умяров Абдулхамит Мустафович; Tatiana N. Bolshakova; Большакова Татьяна Николаевна; Svetlana V. Konstantinova; Константинова Светлана Васильевна; Alexander V. Grishin; Гришин Александр Владимирович; Alexander M. Lyaschuk; Лящук Александр Михайлович; Vladimir G. Lunin; Лунин Владимир Глебович

doi:10.36233/0372-9311-732

Application of the GW module for the immobilization of the red fluorescent protein RFP on peptidoglycan from lactic acid bacteria Lactococcus lactis and Lactobacillus acidophilus

Authors: Dobrynina O.Y.¹, Umyarov A.M.¹, Bolshakova T.N.¹, Konstantinova S.V.¹, Grishin A.V.¹, Lyaschuk A.M.¹, Lunin V.G.¹
Affiliations:
1. The Honorary Academician N.F. Gamaleya National Research Center for Epidemiology and Microbiology
Issue: Vol 103, No 1 (2026)
Pages: 58-65
Section: ORIGINAL RESEARCHES
URL: https://microbiol.crie.ru/jour/article/view/18903
DOI: https://doi.org/10.36233/0372-9311-732
EDN: https://elibrary.ru/ISRLFS
ID: 18903

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Abstract

Introduction. Peptidoglycans from lactic acid bacteria are a safe platform for surface display systems of heterologous proteins for medical purposes. The binding of target proteins to peptidoglycans can occur with the participation of the GW protein module.

Aim: to study the ability of GW module to bind proteins to peptidoglycan derived from lactic acid bacteria.

Materials and methods: The fused protein GW-RFP was obtained with the help of genetically engineering methods. The protein was isolated and purified. Lactic acid bacteria peptidoglycans were isolated and used in the binding reaction with the GW-RFP protein. To monitor the reaction progress, light and fluorescence microscopy and polyacrylamide gel electrophoresis electrophoresis (PAGE) were used.

Results: Methods for isolation and purification of peptidoglycans from Lactococcus lactis and Lactobacillus acidophilus have been developed. The recombinant GW-RFP protein consisting of the red fluorescent protein RFP and the GW module the AltA protein of Staphylococcus aureus has been cloned and expressed in Escherichia coli. It has been shown that the GW-RFP protein binds to the peptidoglycans from L. lactis and L. acidophilus in the amount of 24.9 ± 3.7 μg protein/100 μg (w/w) peptidoglycan from L. lactis and 21.3 ± 3.3 μg protein/100 μg (w/w) peptidoglycan from L. acidophilus. The GW-RFP protein can be removed from the peptidoglycans using a 0.5 M NaCl solution.

Conclusion: The GW module can be used for protein immobilization on the peptidoglycan from both lactococci and lactobacilli.

Keywords

Lactic acid bacteria, Lactococcus lactis, Lactobacillus acidophilus, peptidoglycan, red fluorescent protein RFP, GW module

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Introduction

Lactic acid bacteria are a group of Gram-positive bacterial species that convert carbohydrates into lactic acid. These bacteria are used in the food industry for the fermentation and preservation of various dairy, meat, and vegetable products. They have been granted the GRAS (generally recognized as safe) status, which means that they are generally recognized as non-pathogenic to humans and animals. Due to this, lactic acid bacteria themselves are used as safe platforms for surface display systems of heterologous proteins for medical use [1].

Peptidoglycan (PG) of the cell wall is a unique structure found only in bacteria. It gives rigidity to the bacterial cell, protects it from the environment, determines its shape, and serves as an anchor for various proteins and polymers. The PG scaffold is formed by a glycan heteromer consisting of N-acetylglucosamine and N-acetylmuramic acid residues. The mesh structure of the glycan chains is cross-linked by short peptide bridges, the composition of which depends on the type of bacteria [2]. The main difference between the cell walls of Gram-positive and Gram-negative bacteria, apart from single- and double-layered membranes, is the thickness of the PG layer — in Gram-positive bacteria, this layer is 20–80 nm thick, while in Gram-negative bacteria, it is only 2–7 nm thick [3]. The unique composition of PG, high content of D-amino acids in peptide bridges, and unusual amide bridges make PG an indicator of the presence of bacteria for eukaryotic cells. The immunogenic activity of PG has been characterized for plants and animals [4, 5].

A method for treating lactic acid bacteria has been proposed, resulting in the formation of a PG scaffold from the bacterial cell that retains the shape of the original bacteria [6]. These bacteria-like particles can expose various heterologous proteins on their surface, which are bound to the PG skeleton in one way or another. Such inexpensive, safe and easy-to-manufacture structures can be used to create vaccines, purify antigens, immobilize enzymes, and much more [1].

PG as adjuvants for subunit vaccines have the following properties: they induce mucosal immunity by stimulating IgA production, thus creating protection at the entry point of infection; they activate innate immunity through the TLR-2 receptor. Due to the preservation of the shape of the original bacteria, PG isolated in this way are actively absorbed by dendritic cells and macrophages, which promotes the maturation of dendritic cells and increases the expression of CD80, CD86, and the MHC-II histocompatibility complex [1, 5, 7].

More than 40 different vaccines based on PG particles are currently being developed against bacterial, viral, and parasitic infections caused by Streptococcus pneumoniae, Yersinia pestis, Campylobacter jejuni, Klebsiella pneumoniae, HIV, influenza virus, Newcastle disease virus, MERS-CoV, respiratory syncytial virus, hepatitis E virus, rotavirus, Plasmodium berghei, and Plasmodium falciparum [1].

The exposure of proteins on the surface of bacterium-like particles is used to elicit an immune response (antigen display, antibody display), produce vaccines and alter metabolism (enzyme display) [1, 5].

Target proteins bind themselves to PG in two main ways: covalently or non-covalently. Non-covalent binding of proteins to the cell wall, due to ionic interactions, is carried out with the participation of certain protein domains present in the target proteins [8].

One such domain is the GW module [9]. This module consists of 7 β-strands, 5 of which are folded into an open barrel-shaped conformation. The conservative GW dipeptide (glycine-tryptophan) is located in the fourth β-strand. The length of the GW module is approximately 80 amino acid residues, and the isoelectric point (pI) of this module is about 10, so it has an affinity primarily for acidic ligands (lipoteichoic acid, heparin, protein receptor gClq-R) [10]. However, it is assumed that when teichoic and lipoteichoic acids are unavailable, proteins fused with the GW module can bind to PG [11, 12]. The GW module of the Listeria monocytogenes Ami protein mediates binding to eukaryotic Caco-2 and Hep G2 cells [10, 13]. Several surface proteins of Gram-positive bacteria with GW modules have been identified (autolytic enzymes of S. pneumoniae, amidase of Staphylococcus saprophyticus [14], autolysin of S. aureus [15], amidase, PG hydrolase, and L. monocytogenes internalin [16, 17]. Proteins with GW modules are not only immobilized on the cell surface but can also be partially secreted into the environment. GW modules provide an evolutionarily adapted mechanism that ensures flexibility in the localization and functionality of surface proteins [18].

The aim of this study was to investigate the possibility of using GW modules to bind proteins to PG of lactic acid bacteria. For this purpose, PGs were obtained from Lactococcus lactis and Lactobacillus acidophilus bacteria. A constructed recombinant model protein consisting of the GW module of the AtlA S. aureus protein and the mRFP1 red fluorescent reporter protein was used as the target protein.

Materials and methods

Bacterial strains and media

To isolate PG, cultures of lactic acid bacteria L. lactis subspecies lactis AA119 and L. acidophilus AA141 from the collection of the N.F. Gamaleya Research Center were used. The bacteria were grown on MRS medium (Mann-Rogosa-Sharpe medium; Himedia) or in BHI broth (Brain-Heart Infusion; Himedia) with 2% glucose added at 37°C without aeration. GW-RFP protein cloning was performed in E. coli BL21(DE3) strain with pREP4 plasmid. To obtain the product, E. coli was grown in a special medium for autoinduction [19].

Obtaining peptidoglycan preparations

L. lactis and L. acidophilus cells were processed according to the method described in [6], with modifications [20]. The bacteria were grown to the stationary phase, collected by centrifugation (10 min, 8000g), washed with water, and the wet weight of the cells was determined. The resulting mass was suspended in a 0.25% solution of sodium dodecyl sulfate (SDS) at a ratio of 10 ml of solution per 1 g of raw cell mass. This suspension was kept in a boiling water bath for 2 hours with stirring, the cells were washed with water (approximately 3–4 times until foaming ceased, precipitated for 10 min at 13,000g). The precipitate was suspended in 10% trichloroacetic acid at a rate of 2 ml per 100 mg of wet weight of the precipitate, and this suspension was kept in a boiling water bath for 30 minutes with stirring. The mixture was cooled and washed with water to the pH of the water used for washing.

The resulting precipitate was suspended in 0.1 N HCl at a rate of 3 ml per 100 mg of precipitate. This suspension was also kept in a boiling water bath for 30 minutes with stirring, cooled, and washed with water to the pH of the water used for washing. PG precipitation during washing was carried out by centrifugation at 13,000g for 10 min.

The resulting PG precipitate was weighed and suspended in 10 mM phosphate buffer pH 7.0 at a ratio of 100 mg of raw precipitate weight to 1 mL of buffer. The resulting PG preparation in 10 mM phosphate buffer pH 7.0 with sodium azide (200 μg/mL) was stored in a refrigerator (without freezing).

Design and expression of GW-RFP protein

The nucleotide sequence encoding the GW module of Atl S. aureus ATCC 29213 [9] was synthesized by Eurogen. The source of the mRFP1 fluorescent protein gene was the pTEasy-RFP plasmid from the collection of the N.F. Gamaleya Research Center.

The gene encoding the GW-RFP fusion protein with a hexahistidine tag at the N-terminus was assembled using standard genetic engineering methods and cloned into the pQE13 plasmid, in which the T5 promoter was replaced with the T7 promoter. The resulting plasmid was transformed into the E. coli BL21(DE3) strain carrying the pREP4 plasmid. The producer cells were collected by centrifugation at 7000 g for 15 min.

To isolate the protein, the biomass was resuspended in 10 times the volume of lysis buffer pH 8.0 (50 mM imidazole, 50 mM NaCl, 0.1% Triton X-100, 1 mM MgCl₂) with 150 μg/ml lysozyme and 40 IU/ml benzonase [21], and incubated at room temperature for 30 min. The suspension was treated with ultrasound on a Bandelin Sonopuls HD3200 disintegrator (Bandelin) at 70% amplitude for 5 min. The lysate was then centrifuged at 10,000g and 4°C for 30 min, the supernatant was applied to a column with Workbeads 40 Ni-NTA sorbent (Bio Works), equilibrated with buffer A (50 mM imidazole, 150 mM NaCl, pH 8.0), washed with buffer A with 0.1% Triton X-100 added, then with buffer A with 1 M NaCl added, and with buffer A to remove NaCl. The GW-RFP protein was eluted with buffer (700 mM imidazole, 150 mM NaCl, pH 8.0) and dialyzed against distilled water. The protein concentration was 5.3 mg/mL. The solution of the target protein obtained was stored at –18°C.

GW-RFP protein binding reaction with peptidoglycan

To 400 μg of raw PG weight suspended in 10 mM phosphate buffer pH 7.0, 100 μg of GW-RFP protein from an aqueous solution was added, and the volume of the reaction mixture was brought to 300 μL with the same phosphate buffer (10 mM, pH 7.0) and incubated for 30 min at room temperature with gentle stirring. The mixture was then centrifuged for 10 min at 13,000g, and the precipitate was resuspended in 40 µL of the same buffer. The resulting precipitate was analyzed by polyacrylamide gel electrophoresis (PAGE) using a standard method. PG samples subjected to the same manipulations but without the addition of GW-RFP protein served as controls.

Protein determination

Protein and peptide concentrations were determined using the Lowry method.

Light and fluorescence microscopy

For microscopic analysis, the obtained PG preparations were stained using the standard Gram method. For fluorescence microscopy, 5 μL of PG suspension with bound GW-RFP protein was applied to a microscope slide and air-dried. After applying immersion oil for fluorescence, the samples were examined using a Zeiss Axio Imager Z1 microscope (Zeiss) equipped with a Cascade II camera (Photometrics).

Results

Production of peptidoglycan preparations

The treatment of lactic acid bacteria leads to the formation of non-viable particles consisting mainly of the PG scaffold. The non-viability of such particles is proven by the absence of growth when PG drugs are seeded on a solid MRS medium. The micrographs shown in Fig. 1 show that the obtained PG preparations partially retain the shape of bacteria (empty shells are visible), but PG is stained with gentian violet less intensely than the original bacteria. The amount of peptides in PG was determined using the Lowry method; the values are given in the table. Fig. 2 shows an electrophoregram of the obtained PG drugs in polyacrylamide gel; empty lanes prove the absence of protein molecules released during SDS treatment. However, a noticeable amount of peptides is detected in PG preparations (Table).

Fig. 1. Lactic acid bacteria and PGs obtained from them (Gram staining).

a — L. lactis; b — PGs from L. lactis; c — L. acidophilus; d — PGs from L. acidophilus.

Fig. 2. Electropherogram of the GW-RFP protein binding to PGs of L. lactis and L. acidophilus (Coomassie staining).

1 and 2 — PGs of L. lactis (1) and L. acidophilus (2) before protein binding; 3 and 4 — precipitates after the binding of PGs of L. lactis (3) and L. acidophilus (4) with the GW-RFP protein; 5 and 6 — supernatants after the binding of PGs of L. lactis (5) and L. acidophilus (6) with the GW-RFP protein; 7 and 8 — precipitates of PGs of L. lactis (7) and L. acidophilus (8) after the binding with the GW-RFP protein and incubation in the presence of 0.5 M NaCl; 9 and 10 — supernatants after the binding of PGs L. lactis (9) and L. acidophilus (10) with GW-RFP protein and incubation in the presence of 0.5 M NaCl; 11 — GW-RFP protein.

Binding of GW-RFP protein to PG of L. lactis and L. acidophilus

Sample	Lactococcus lactis		Lactobacillus acidophilus
Sample	precipitate (μg of peptides per 100 μg of PG)	supernatant (μg of protein in 300 μL of 10 mM phosphate buffer, pH 7.0)	precipitate (μg of peptides per 100 μg of PG)	supernatant (μg of protein in 300 μL of 10 mM phosphate buffer, pH 7.0)
PG	16 ± 5	0	15.2 ± 1.7	0
PG bound to GW-RFP protein	24.9 ± 3.7	0	21.3 ± 3.3	0
PG bound to GW-RFP protein and then treated with 0.5 M NaCl solution	0	118.2	0	106

GW-RFP marker protein

In this study, the GW module from the Alt S. aureus protein was fused with the target protein, the red fluorescent protein mRFP1. The amino acid sequence of this recombinant protein is as follows:

5–10 amino acid residues (a.r.) — hexahistidine tag; 11–174 a.r. — GW module; RFP protein — 175–400 a.r.

An aqueous solution of the resulting recombinant GW-RFP1 protein (with a molecular weight of 44.5 kDa) has a dark pink color. Its intensity depends on the concentration of the protein in the solution [22].

GW-RFP protein binding reaction with peptidoglycan

The ability of the GW module to take part in the binding of proteins to PG of lactic acid bacteria was tested in a binding reaction. The degree of binding of the recombinant protein to PG was judged by the appearance of a dark pink precipitate after separation of the supernatant at the end of the reaction. Furthermore, the amount of protein in the precipitate and supernatant was tested using both electrophoresis (Fig. 2) and the Lowry protein assay.

The electropherogram (Fig. 2) shows that the protein bound to PG corresponds to the GW-RFP protein.

It was assumed that the protein bound to PG would remain in the precipitate, while the unbound protein would be found in the supernatant. We were able to determine the amount of GW-RFP protein that binds to a certain amount of PG. It turned out that approximately 0.56 nmol of GW-RFP protein binds to 100 μg of raw weight PG L. lactis, and 0.48 nmol binds to 100 μg of raw weight PG L. acidophilus (Table).

It is known from the literature that the degree of protein binding to PG depends on the ionic strength of the solution—the higher it is, the weaker the protein binding [11]. Indeed, the addition of a buffer solution containing 0.5 M NaCl to the PG precipitate of both L. lactis and L. acidophilus with immobilized GW-RFP protein led to protein dissociation and its transition into the supernatant (Table; Fig. 2). Thus, proteins bound to PG using the GW module can be eluted using solutions with high ionic strength.

Storage of PG preparations at 4°C in a buffer solution with sodium azide, lyophilization, autoclaving (15 min, 121°C), freezing (–18°C, 72 h), and thawing did not affect the binding properties of PG (data not shown).

Judging by the fluorescent micrographs (Fig. 3), PG obtained by the method proposed in the study mainly retain the shape of the original lactobacilli (cocci and bacilli), and the binding fluorescent protein GW-RFP forms a luminous halo around the particles.

Fig. 3. Fluorescence micrograph of PGs bound to the GW-RFP protein.

a — PGs from L. lactis; b — PGs from L. acidophilus.

Discussion

It is known from the literature that treatment of Gram-positive bacteria cells with SDS causes loosening of the cell wall, and subsequent heat treatment in the presence of trichloroacetic and hydrochloric acids removes teichoic acids, lipids, most proteins, and DNA [20]. A significant amount of peptides in PG are peptide cross-links of glycan chains (Table). These peptide bridges partially preserve the shape of the cells of the microorganism from which PG is obtained (in our case, cocci and bacilli). The resulting immunologic agents are safe for humans and animals, do not contain live bacteria, and do not require expensive reagents and equipment for their isolation. These immunologic agents can be used to stimulate the innate immune response as vaccine adjuvants.

The Alt S. aureus protein belongs to the adhesins and autolysins and contains a region with several GW (glycine-tryptophan) repeats in its molecule. The presence of these modules allows the microorganism to bind securely to the cell wall of host cells [9]. The fusion of the GW module with the reporter part—the red fluorescent protein RFP—led to the creation of a convenient model that allows one to see the binding of the protein to the PG precipitate by the appearance of a dark pink color of the precipitate. The color of the protein disappears in the presence of SDS, indicating that the preservation of the corresponding conformation of the RFP protein is important for the color to appear. The dark pink color of the precipitate after the protein binds to PG confirms that the immobilization of the model protein using the GW module does not change the initial conformation of the reporter part.

Fluorescent micrographs show that immobilization of the model protein with the GW module occurs on the outer surface of PG particles.

Conclusion

Lactic acid bacteria with GRAS status, i.e., completely safe for humans and animals, were used to obtain PG. PG isolated from these bacteria is convenient for various manipulations and stable in storage.

The most obvious use of PG from lactic acid bacteria is the creation of immunologic agents and vaccines based on their platform. Such agents are economically advantageous, as they are not difficult to produce and provide a strong immune response with a simplified vaccination schedule [1, 7].

The GW module, extracted from its natural context, fuses with target proteins, and thus it is possible to bind the protein of interest to the PG-rich cell wall of Gram-positive bacteria for use as subunit vaccines and obtain a stable and bioavailable drug.

This study shows that a protein containing the GW module binds well to PG obtained from lactic acid bacteria, both lactococci and lactobacilli.

Known GW modules vary greatly in size and amino acid composition, so when creating immunologic agents, it is possible to select the most suitable designs with target proteins to preserve the conformation necessary for a proper immune response and optimal binding to PG of lactic acid bacteria.