RU2593946C1 - METHOD OF EXTRACTING LIPOPOLYSACCHARIDE Chlamydia trachomatis - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry. Disclosed is a method of extracting lipopolysaccharide Chlamydia trachomatis. Method involves extraction of lipopolysaccharide of bacterial cells with 90 % aqueous solution of phenol at 50 °C. Then supernatant is separated from undissolved components by centrifuging, diluted with 0.1 M tris-HCl buffer at a ratio of 1:9. Extraction of lipopolysaccharide Chlamydia trachomatis is carried out by affine chromatography on concanavalin-A-sepharose 4B. Extraction is followed by washing with 1 M NaCl solution, buffered with tris-HCl buffer, and elution with 0.4-0.45 M solution of N-acetyl-D-glucosamine in tris-HCl buffer with further dialysation and lyophilisation.

EFFECT: invention provides high degree of purity of extracted lipopolysaccharide.

1 cl, 4 dwg, 1 tbl

Description

The invention relates to medicine, namely biochemistry, in particular to methods for extracting lipopolysaccharides, and can be used to isolate lipopolysaccharide from the microbial mass of Chlamydia trachomatis.

Chlamydia trachomatis is considered the most common causative agent of bacterial sexually transmitted infections [Kalinina SN, Tiktinsky OL Treatment of chronic prostatitis due to chlamydial and ureaplasma infection and complicated by male infertility. Urology. 2010; 3: 52-57]. One of the pathogenicity factors of Chlamydia trachomatis, which affects the physiology of reproductive processes in both men and women and leads to the development of infertility associated with the infectious and inflammatory processes of the urogenital tract, are lipopolysaccharides (LPS) - complex macromolecules found in the cell wall of gram-negative bacteria.

It is well known that LPS of Chlamydia trachomatis, even in minimal doses, are powerful endotoxins that can exert a cytotoxic effect on germ cells, inhibit their functions and induce death [Bukharin OV, Kuzmin MD, Ivanov Yu.B. The role of the microbial factor in the pathogenesis of male infertility. Zhurn. microbiol. 2000; 2: 106-110; Hosseinzadeh S., Pacey A.A., Eley A. Chlamydia trachomatis induced death of human spermatozoa is caused primarily by lipopolysaccharide // J. Med. Microbiol. - 2003. - V. 52. - No. 3. - p. 193-200].

The study of bacterial factors such as LPS is crucial in light of the search for new approaches to the treatment and prevention of chronic infections. The correct selection of the method of isolation and good purification of LPS guarantees the successful determination of its structural centers, which determine the biological activity of macromolecules as specific antigens, endotoxins, and pharmacological preparations.

For the successful determination of biological functions, chemical composition and structure, serological activity of lipopolysaccharide (LPS), there are a number of methods for its isolation and purification, but all of them are not without drawbacks.

In practice, the water-phenolic method for isolating LPS from gram-negative bacteria cells is well known [Westphal O., Luderitz O., Bister F. Uber die Extraktion von Bakterien mit Phenol / Wasser // Z. Naturforsch., 1952. No. 3. - P. 148-155; Westphal O., Jann K. Bacterial lipopolysaccharides. Extraktion with phenol-water and further applications of the procedure. - Methods Carbohydr. Chem., 1965, No. 5, p. 83-91], including processing the microbial mass with a water-phenolic mixture at 65 ° C for 45 minutes After cooling, the mixture was separated by centrifugation. The aqueous phase is sucked off and dialysed for 3-4 days against distilled water to remove phenol and low molecular weight impurities. LPS is precipitated by ultracentrifugation at 100,000 g and 15 ° C for 3 hours.

The disadvantages of this method are:

- insufficient purity of the LPS preparation;

- Duration: the need for prolonged dialysis and subsequent additional purification of the drug by ultracentrifugation;

- the use of expensive equipment (ultracentrifuge);

- low yield due to the loss of LPS at the stage of ultracentrifugation.

There is another way to isolate LPS from the R-forms of gram-negative bacteria using the phenol-chloroform-petroleum ether extraction method [Galanos C., Luderitz O., Westphal O.A. New method for the extraction of R lipopolysaccharide // Eur. J. Biochem. - 1969. - V. 9, No. 2. - P. 245-249], in which dry bacterial cells are suspended three times in a mixture of 90% phenol: chloroform: petroleum ether (mp. 40-60 ° C) in a ratio of 2: 5: 8, respectively, shaken for 2 minutes with cooling (not higher than 20 ° C) and centrifuged for 15 minutes at 5000 rpm. The supernatants are combined, chloroform and petroleum ether are removed by distillation at 30-40 ° C. Distilled water is added dropwise to the phenolic phase until the LPS precipitates, which is separated by centrifugation at 3000 rpm for 10 minutes, and then the precipitate is washed 2-3 times with small portions of 80% phenol, washed three times with ether to remove phenol and dry in vacuo. LPS is dissolved in 50 ml of water, heated to 45 ° C, the viscosity of the solution is reduced by placing it in an ultra-vibrator for 5 min, after which LPS is precipitated by ultracentrifugation at 100,000 g for 4 hours. The precipitate is separated and dissolved in a small amount of water and lyophilized.

To isolate LPS of Chlamydia trachomatis, the Galanos C. method described above is used, as well as the method modified by Nurminen M. [Nurminen M., Rietschel ET, Brade H. Chemical characterization of Chlamydia trachomatis lipopolysaccharide // Infect Immun. - 1985. - V. 48. - p. 573-575], which consists in the fact that the dry cells of Chlamydia trachomatis (elementary bodies) are suspended in 2.5 ml of a 90% aqueous phenol solution and homogenized for 5 min, followed by stirring on a magnetic stirrer at 50 ° C for 30 min To the mixture was added 4 ml of petroleum ether (mp. 90-100 ° C) and 2.5 ml of chloroform and stirred for 1 h at room temperature. After centrifugation, the supernatant was freed from organic solvents under reduced pressure and the remaining phenolic phase was precipitated with 10 volumes of chilled acetone (-20 ° C). The precipitate is washed three times with cold acetone, dissolved in water and lyophilized. The dry material is re-extracted with a mixture of phenol-chloroform-petroleum ether according to the method of Galanos C. (see above), precipitated with water, washed with acetone, dissolved in water and dried under vacuum.

The disadvantages of the methods Galanos S. and Nurminen M. are:

- the systems of organic substances used to isolate LPS affect its structure, conformation, lead to partial degradation of the polymer, which is extremely undesirable when conducting molecular biological studies, since these changes affect the biological activity of endotoxin;

- organic substances used to isolate LPS are volatile, aggressive, toxic to humans and environmentally harmful substances.

- multi-stage and duration of isolation procedures, which complicates the process of obtaining pure LPS.

- a low degree of purification of the obtained LPS preparations from impurities of bacterial components;

- use of special and expensive equipment;

- low product yield due to the loss of LPS at the stages of ultracentrifugation or at several freeze drying procedures, which can also significantly violate the native properties of LPS (first of all, the solubility of the target product worsens).

Closest to the claimed method is a prototype - a method for the isolation of LPS Chlamydia trachomatis (serovar E and LGV) [Hosseinzadeh S., Pacey AA., Eley A. Chlamydia trachomatis induced death of human spermatozoa is caused primarily by lipopolysaccharide // J. Med. Microbiol. - 2003. - V. 52. - No. 3. - p. 193-200] by suspending dry bacterial cells in 2.5 ml of a 90% aqueous phenol solution, homogenizing for 5 minutes, followed by stirring on a magnetic stirrer at 50 ° C for 30 minutes. To the mixture was added 4 ml of petroleum ether (mp. 90-100 ° C) and 2.5 ml of chloroform and stirred for 1 h at room temperature. After centrifugation at 10,000 g for 30 min, the supernatant was freed from organic solvents on a rotary evaporator at 40 ° C, and the remaining phenolic phase was precipitated with 10 volumes of chilled acetone (-20 ° C). The precipitate was washed with cold acetone and dried in vacuo.

However, this method of isolating LPS of Chlamydia trachomatis has the following disadvantages:

- refers to harsh chemical methods for the isolation of endotoxin, which leads to a change in the initial molecular organization of the biopolymer, violating its native structure and biological properties;

- the use of volatile, aggressive, toxic to humans and environmentally harmful substances;

- unsatisfactory quality of the obtained LPS preparation due to the low degree of its purification due to the presence of other bacterial components in the preparation, in particular a large amount of protein and nucleic acids, which may complicate its further use;

The invention is aimed at increasing the degree of purity, i.e. improving the quality of LPS Chlamydia trachomatis.

The specified technical result is achieved by the fact that after phenol extraction of the lipopolysaccharide from bacterial cells in the next step, the supernatant is separated from the insoluble components by centrifugation for 15 min at 5000 rpm and diluted with 0.1 M Tris-HCl buffer (pH 7.5) in 1: 9 ratio, and at the final stage of isolation, affinity chromatography of Chlamydia trachomatis lipopolysaccharide on concanavalin A-sepharose 4B is carried out, then washed with 1 M NaCl solution, buffered Tris-HCl buffer, and bound on a lipopolysakh column is eluted rid 0.4-0.45 M solution of N-acetyl-D-glucosamine in Tris-HCl buffer with pH = 8.0, with further dialysis and lyophilization.

The isolation of LPS of Chlamydia trachomatis was carried out as follows. Dry Chlamydia trachomatis bacteria were used as the source of LPS, which were suspended in 2.5 ml of a 90% aqueous phenol solution, homogenized for 5 min, and stirred on a magnetic stirrer at 50 ° С for 30 min. The undissolved components were removed by centrifugation for 15 min at 5000 rpm, the supernatant was diluted with 0.1 M Tris-HCl buffer (pH 7.5) in a 1: 9 ratio.

The final stage of the isolation of LPS of Chlamydia trachomatis was affinity chromatography on concanavalin A-sepharose 4B. The entire volume obtained in the previous fractionation step was passed through a concanavalin A Sepharose column equilibrated with Tris-HCl buffer (pH 7.5). From non-specifically bound proteins, the column was washed with 1 M sodium chloride buffered with Tris-HCl buffer. Bound on a LPS column was eluted with a stepwise gradient of the concentration of N-acetyl-D-glucosamine from 0.1 to 0.5 M in Tris-HCl buffer (pH = 8.0). Fractions were collected, in each of which the LPS was determined by reaction with phenol and sulfuric acid, RNA by absorption at 260 nm, protein by Lowry. The maximum LPS concentration was released upon elution with a 0.4-0.45 M solution of N-acetyl-D-glucosamine in Tris-HCl buffer (pH = 8.0). Fractions containing LPS were collected, dialyzed against distilled water, lyophilized and stored at -20 ° C.

To confirm the successful isolation of LPS of Chlamydia trachomatis, 20 μl of the LPS sample obtained by the proposed method was added to a 14% polyacrylamide gel (PAGE) and electrophoresis was performed at room temperature in Tris-glycine buffer (pH 8.3) at a voltage of 100-150 V LPS of E. coli (serotype 055: B5, Sigma) was used as a positive control. To detect LPS, gels were treated with silver nitrate (Fig. 1.).

In fig. 1 (SDS page electrophoresis), where lane 1 — LPS isolated from Chlamydia trachomatis (serovar E) in a patented manner; lane 2 — LPS of E. coli (serotype 055: B5, “Sigma”), it is seen that after affinity chromatography, the LPS preparation of Chlamydia trachomatis is homogeneous with electrophoretic purity control.

The proposed method was successfully tested at the Department of Chemistry SBEI HPE Astrakhan State Medical University of the Ministry of Health of Russia during 2014.

Purified Chlamydia trachomatis elementary bodies (ETs) were obtained from McCoy cells according to the method of [Caldwell N.D., Kromhout J., Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis // Infect Immun. - 1981. - V. 31. - p. 1161-1176]. LPS was isolated from lyophilized cells (ET) of Chlamydia trachomatis (Serovar E).

The following is the result of testing.

50 mg of dry cells of ET Chlamydia trachomatis were suspended in 2.5 ml of a 90% aqueous phenol solution, homogenized for 5 min and stirred on a magnetic stirrer at 50 ° C for 30 min. The undissolved components were removed by centrifugation for 15 min at 5000 rpm, the supernatant was diluted with 0.1 M Tris-HCl buffer (pH 7.5) in a 1: 9 ratio.

The final stage of the isolation of LPS of Chlamydia trachomatis was affinity chromatography on concanavalin A-sepharose 4B. The entire volume obtained in the previous fractionation step was passed through a concanavalin A Sepharose column equilibrated with Tris-HCl buffer (pH 7.5). From non-specifically bound proteins, the column was washed with 1 M sodium chloride buffered with Tris-HCl buffer. Bound on a LPS column was eluted with a 0.4-0.45 M solution of N-acetyl-D-glucosamine in Tris-HCl buffer with pH = 8.0. Fractions containing LPS were collected, dialyzed against distilled water, lyophilized and stored at -20 ° C.

As a result, an LPS preparation of Chlamydia trachomatis isolated by the patented method (LPS ₁ ) was obtained and characterized, and for comparison, an LPS preparation of Chlamydia trachomatis obtained by the prototype method (LPS ₂ ) was taken.

LPS preparations, isolated in different ways, are white flakes, readily soluble in water, sucrose-phosphate buffer solution or in 0.9% NaCl solution.

The chemical composition was determined by conventional methods: total carbohydrates according to Dubois with thymol and sulfuric acid, proteins according to Lowry, nucleic acids - according to Spirin.

A comparative analysis of the preparations isolated by the patented method (LPS ₁ ) and the prototype method (LPS ₂ ) showed similar characteristics in terms of the percentage of the yield of samples from the dry weight of the cells (2-3%), however, there was a significant difference in other parameters (Table 1) . Thus, the LPS ₂ sample contained an increased amount of 6.2% protein and 3.5% nucleic acids, i.e. was not sufficiently purified.

Table 1 shows a comparative characteristic of the purity of the LPS of Chlamydia trachomatis obtained by the proposed isolation method (LPS ₁ ) and the prototype method (LPS ₂ ).

To confirm the isolation and purity of LPS of Chlamydia trachomatis obtained by the proposed method, reverse phase high performance liquid chromatography (RP HPLC) was performed on a Gilson chromatographic complex.

RP-HPLC was performed on a S-18 silosorb sorbent (Czech Republic), which is silica gel particles coated with a monomolecular layer of octadecyltrichlorosilane. The mobile phase was a linear gradient of acetonitrile in a 0.1% trifluoroacetic acid solution.

For analysis, we used: a sample of the initial LPS preparation of Chlamydia trachomatis isolated after phenolic extraction from bacterial cells (LPS ₀ ) (Fig. 2); a sample of the LPS preparation of Chlamydia trachomatis isolated by the patented method (LPS ₁ ) (Fig. 3); a sample of the LPS preparation of Chlamydia trachomatis isolated by the prototype method (LPS ₂ ) (Fig. 4).

Analysis of the chromatograms of samples LPS ₀ , LPS ₁ and LPS ₂ by RP HPLC (Figs. 2, 3, 4, respectively) showed the main peak, which contains the polysaccharide. A comparative analysis of the chromatograms of the LPS samples of Chlamydia trachomatis shows that the LPS ₀ preparation is not purified; in the LPS ₁ preparation obtained by the patented method, there is only one main peak, which proves the purity of the drug. In contrast to LPS _1, in the LPS ₂ preparation obtained by the prototype method, 3-4 additional significant peaks are noted at 15.4; 17.8; 23.3 and 31 minutes

When calculating the purity of the LPS of Chlamydia trachomatis by the planimetric method in the LPS ₀ preparation, the proportion of LPS according to RP HPLC was approximately 8%; in the LPS ₁ preparation isolated by the patented method, 94.7%; and in the LPS ₂ preparation - 77.6%.

The main advantage of our proposed method for isolating LPS of Chlamydia trachomatis is the use of affinity chromatography on concanavalin A-sepharose. Affinity chromatography is the most appropriate and rational method for obtaining pure preparations, the advantages of which are also a small number of operations and a high yield of product. The effective use of carbohydrate-binding proteins, lectins, as ligands in affinity chromatography is widely known. For example, the use of lectins mainly for the purification of a wide variety of glycoproteins significantly simplifies and speeds up the process of purification of the latter.

The most important characteristic of lectins is their property of reversibly and selectively binding carbohydrates without causing their chemical conversion, the specificity of their interaction with carbohydrates. Lectins interact with both free mono- and oligosaccharides, as well as with carbohydrate residues in glycoproteins, polysaccharides and glycolipids. Considering the carbohydrate specificity of concanavalin-A lectin and the presence of D-glucosamine in the LPS composition of Chlamydia trachomatis, their effective binding is observed. This allows you to once increase the degree of purification of LPS (compared with the prototype method).

The advantages of the patented method for the isolation of LPS of Chlamydia trachomatis using affinity chromatography on concanavalin A-sepharose, in that the method allows to improve the quality of the LPS preparation of Chlamydia trachomatis compared to the prototype method and to obtain a more chemically pure and homogeneous LPS, which simultaneously makes it possible to reduce the use of toxic to humans and environmentally harmful substances.

A higher quality of the LPS preparation isolated by the described method will allow using the drug in a wide range of microbiological, biochemical and immunological studies.

Claims (1)

The method of isolation of Chlamydia trachomatis lipopolysaccharide by extracting it from bacterial cells with a 90% aqueous phenol solution at 50 ° C, characterized in that in the next step the supernatant is separated from the insoluble components by centrifugation for 15 min at 5000 rpm and diluted with 0, 1 M Tris-HCl buffer (pH 7.5) in a 1: 9 ratio, and at the final stage of isolation, affinity chromatography of Chlamydia trachomatis lipopolysaccharide on concanavalin A-Sepharose 4B is carried out, then washed with 1 M NaCl solution, buffered Tris-HCl buffer and elute the lipopolysaccharide bound to the column with a 0.4-0.45 M solution of N-acetyl-D-glucosamine in Tris-HCl buffer with pH = 8.0 with further dialysis and lyophilization.

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