RU2514662C2 - Method for in vitro determination of activation of plasminogen with bacteria - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to medical microbiology and a method of determining activation of plasminogen with bacteria. The method involves adding protamine sulphate to a prepared supernatant fluid, incubating the obtained mixture, depositing cells by centrifuging, incubating the supernatant fluid with the protamine sulphate, depositing protein and detecting activation of plasminogen with bacteria from the amount of split arginine, content of which is determined by Sakaguchi method from the red colour of the sample.

EFFECT: invention enables to detect activation of plasminogen with bacteria in vitro using protamine.

4 tbl, 4 ex

Description

The present invention relates to the field of medical microbiology and can be used in applied and basic research related to the study of the subtle mechanisms of pathogenesis, invasion, bacteremia and dissemination of microorganisms.

It is known that a number of microbial pathogens use the human plasminogen / plasmin system to overcome tissue barriers during the so-called bacterial metastases (1). They express receptors on their surface for fixation and subsequent activation of plasminogen under the influence of urokinase (uAP) and tissue plasminogen activator (tAP) of the host; others use their own proteases belonging to the family of optins. Thus, the activation of plasminogen in plasmin significantly enhances the pathogenetic capabilities of the pathogen by enhancing its destructive potential, aimed at scrapping the innate protective forces of the sensitive host. Therefore, a convenient and safe method for detecting plasminogen activation by one or another pathogen is relevant.

A known method of plasminogen activation by Yersinia pestis KIM D34 cells according to Haiko J. (2), which consists in the fact that the chromogenic substrate Val-Leu-Lys-p-nitroaniline dihydrochloride (S-2251) is used to record plasmin activity, which is added to the incubation mixture . The formation of plasmin is recorded using a special reader for tablets at 405 nm.

However, the known synthetic chromogenic substrate is characterized by instability during storage, the duration of the process (since the incubation sometimes takes 24 hours), the cost of the reagents and requires special equipment.

For the prototype, a method for determining plasminogen by a chromogenic substrate was selected (see A. P. Mamot, A. I. Mamaev, Z. S. Barnaza and others. “Method for determining plasminogen by a domestic chromogenic substrate and its diagnostic value”, Journal of Clinical Laboratory Diagnostics, No. 3, 2000, pp.21-23) (3), which consists in the fact that to 0.5 ml of a suspension of bacteria in physiological saline (10 ⁹ cells / ml) add 15 μg of human plasminogen and 30 μl of chromogenic substrate For- Ala-Phe-Lys-pNa. HBr in an amount of 4 μg / ml in a final volume of 540 μl, then incubated at 37 ° C for 0.5-24 hours, cleavage of the substrate with plasmin was recorded at 405 nm by cleaved pNa and expressed in extinction numbers ΔA ₄₀₅ = t ₁ -t ₀ , as a result of hydrolysis of the substrate a colored substance is formed, confirming the presence of plasmin.

The disadvantage of the prototype is the complexity and duration of the research process, especially since the synthesis of the commercial preparation For-Ala-Phe-Lys-pNa itself. HBr is a laborious multi-step process involving 7 stages.

In addition, the registration of the ability of the protease of the outer membrane proteins of pathogens of especially dangerous infections to activate plasminogen by cleaving chromogenic substrates is difficult due to the peculiarities of working with especially dangerous infections.

The technical task of the invention is the development of a simple and more time-efficient way of recording the phenomenon of plasminogen activation by bacteria of especially dangerous infections in vitro under conditions that are as safe as possible for the experimenter.

This object is achieved in that in the known method for determining plasminogen activation in bacteria in vitro, including the reaction of interaction of plasmin with a peptide substrate, protamine sulfate is used as the latter, which is added in an amount of 100 μl to the prepared supernatant, which is prepared as a mixture: to 0.49 ml of 0.2 M phosphate buffer, pH 8.0, add 0.5 ml of a suspension of bacteria in physiological saline (5 × 10 ⁹ cells / ml) and 15 μg of human plasminogen in a final volume of 1 ml, then the resulting suspension is incubated in t 1 hour at 37 ° C and precipitated by centrifugation at 11000 rpm for 5 minutes, after which the samples were incubated for 2 hours at 37 ° C, then the protein in the samples was precipitated by adding 1 ml of 20% TCA, placed in an ice bath and the activation was recorded plasminogen by bacteria by the amount of cleaved arginine, the content of which is determined by the Sakagushi method by staining the samples in red, the brighter the color, the greater the amount of cleaved arginine, and correspondingly higher activity of the studied strain.

The method is as follows. To confirm the operation of the method were used strains of microorganisms V. cholerae (5879, 17259, 17779, 17625); Y. pestis (EV, TRU, GWR); F.tularensis (Schu, 543, novicidae, 1510) and strains of bacteria E. coli (QD ₅₀₀₃ , QD ₅₀₀₃ pRD13) from the collection of the Rostov-on-Don Antiplague Institute.

Pre-prepare a suspension of bacteria according to the optical standard of turbidity in a concentration (5 × 10 ⁹ cells / ml), which are disinfected by the addition of sodium merthiolate (1: 10000).

Then, an incubation mixture is prepared for the reaction; for this, 0.5 ml of a suspension of bacteria in physiological saline (5 × 10 ⁹ cells / ml) and 15 μg of human plasminogen are added to 0.49 ml of 0.2 M phosphate buffer, pH 8.0 volume of 1 ml. Instead of a suspension of cells, physiological saline (plasminogen control) is added to the first control tube, and physiological saline instead of bacterial suspension and plasminogen (protamine control) is added to the second control tube. Incubation is carried out for 1 hour at 37 ° C, after which the cells are precipitated by centrifugation (11000 rpm, Heareus biofug) for 5 minutes. The supernatant is transferred to a clean tube. 100 μl of protamine is added to the experimental and control samples, which corresponds to 10 mg / ml of the mentioned substrate.

After this, the samples are incubated for 2 hours at 37 ° C. Then the protein is precipitated by adding 1 ml of 20% TCA, thereby achieving additional disinfection of the contents of the sample. The supernatant clarified by centrifugation is transferred to glass tubes, which are placed in an ice bath and used to determine the arginine content by the Sakagushi method (4).

The amount of arginine cleaved by activated plasminogen is determined by the calibration curve, which is built using arginine hydrochloride as a standard. The activity is expressed in μg of arginine cleaved from protamine by plasmin formed as a result of plasminogen activation by cells of the studied microorganism for 1 hour incubation at 37 ° C.

In addition, the method is accompanied by staining the samples in red, the brighter the color, the higher the arginine content and therefore the higher the activity of the studied strain.

Example No. 1.

The cultures of V. cholerae 5879, 17259 (isolated from patients and carrying the intact ompT gene that determines the plasminogen-activating ability of the strains) and 17625, 17779 (isolated from water samples and lacking the intact ompT gene as a result of mutational events) were used as the studied strains.

As can be seen from table 1, strains of cholera vibrios obtained from clinical material had the ability to activate human plasminogen in vitro, as indicated by high arginine content and staining of samples, while strains isolated from water samples did not have this ability.

Example No. 2.

The experimental strains E. coli QD ₅₀₀₃ , E. coli QD ₅₀₀₃ pRD13 (with the plasmid carrying the plasminogen activator gene pla ⁺ Yersinia pestis) were taken for study.

As follows from Table 2, E. coli strains had plasminogen-activating ability, and the strain transformed with plasmid pRD13 with the pla gene from the plague microbe had 2 times higher activity than the initial one due to the summation of the activities of the two genes.

Example No. 3.

The strains of the tularemia causative agent, Francisella tularensis Schu, 543 (virulent strains), 1510 and F. novicidae (avirulent strains) were used as the studied strains (Table 3).

From the data of table 3 it can be concluded that virulent strains had a high ability to activate plasminogen, while non-pathogenic for humans variants were reduced.

Example No. 4.

As the studied strains, cultures of the plague pathogen were used - Yersinia pestis EV (pla +, vaccine strain), TRU, GEM (Δpla, avirulent strains) (Table 4).

From the data of table 4 it can be seen that the strain carrying the pla gene has a high ability to activate plasminogen, while strains that do not contain pla in their genome did not possess this activity.

From examples 1-4 it follows that the claimed method allows to characterize bacteria by plasminogen-activating ability, not inferior to the method described in the closest analogue, subject to the rules of anti-epidemic protection of the experimenter.

Thus, plasminogen activation is recorded by bacteria using protamine sulfate, which is 60% composed of arginine residues, the bonds between which plasmin cleaves, as a substrate.

The use of the present invention allows using protamine simple, not requiring expensive reagents and equipment, as well as time-efficient (within 4 hours), safe for the experimenter conditions to record the phenomenon of plasminogen activation by bacteria in vitro.

In the future, the proposed method can be used in the study of the pathogenesis of cholera, tularemia and other infections, as well as in the development and characterization of vaccines from causative agents of especially dangerous infections based on proteins of the outer membrane and vesicles from them.

Information sources

1. Lahteenmaki K., Edelman S., Korhonen T.K. Bacterial metastasis: the host plasminogen system in bacterial invasion // Trends in Microbiology. - 1984 - Vol.13. - N2. - P.79-85.

2. Haiko J., Suomalainen M., Ojala T., Lahteemaki K., Korhonen T.K., Breaking barriers - attack on innate immune defense by omptin surface proteases ofenterobactsrial pathogens. // Innate Immunity 2009. - Vol. 15. - No. 2. - p. 67-80.

3. Momot A.P., Mamaev A.N., Barkagan Z.S., Nevedrova O.E., Makarov V.A., Voyushina T.L., Erin D.M. Method for determination of plasminogen with a domestic chromogenic substrate and its diagnostic value. // Clinical laboratory diagnostics., 2000, No. 3, p.21-24.

4. Gilboe D.D., Williams J.N. Evaluation of the Sakagushi reaction for quantitative determination of arginine. // Proc. Soc. Exp. Biol. Med. - 1956 - Vol. 91. - No. 4. - p. 535-536.

Table 1 No. Strain V. cholerae Protamine Chromogenic substrate μgArg / 10 ⁹ m.k / hour OD ₄₀₅ , A ₄₀₅ = t ₁ -t ₀ (in 24 h) one. 5879 48.0 0.19 2. 17259 22.0 0.153 3. 17779 0 0 four. 17625 0 0

table 2 No. Strain E. coli Protamine Chromogenic substrate μgArg / 10 ⁹ m.k / hour OD ₄₀₅ , A ₄₀₅ = t ₁ -t ₀ (in 24 h) one. QD ₅₀₀₃ 9.22 0.378 2. QD ₅₀₀₃ pRD13 19.83 0.512

Table 3 No. Strain F. Tularensis Protamine Chromogenic substrate mcgarg / 10m ⁹ .k / hour OD ₄₀₅ , A ₄₀₅ = t ₁ -t ₀ (in 24 hours) one. Schu 10.0 0.137 2. 543 5,6 0.128 3. novicidae 2.6 0.155 four. 1510 1.55 0.277

Table 4 No. Strain Y. pestis Protamine Chromogenic substrate μgArg / 10 ⁹ m.k / hour OD ₄₀₅ , A ₄₀₅ = t ₁ -t ₀ (in 24 hours) one. EV 155.19 0.149 2. TRU 0.78 0 3. ZhVR 0 0

Claims (1)

A method for determining plasminogen activation by bacteria in vitro, including the interaction of plasmin with a peptide substrate, characterized in that protamine sulfate is used as a substrate, which is added in an amount of 100 μl to the prepared supernatant, the latter is prepared as a mixture: 0.49 ml 0 , 2 M phosphate buffer, pH 8.0, add 0.5 ml of a suspension of bacteria in physiological saline (5 × 10 ⁹ cells / ml) and 15 μg of human plasminogen in a final volume of 1 ml, then the resulting mixture is incubated for one hour at 37 ° C and cage and precipitated by centrifugation at 11000 rpm for 5 min, after which the supernatant with protamine sulfate was incubated for 2 hours at 37 ° C, the protein was precipitated by adding 1 ml of 20% TCA, placed in an ice bath, plasminogen activation by bacteria was recorded by the amount of cleaved arginine, the content of which is determined by the Sakagushi method by staining the samples in red, the brighter the color, the higher the arginine content.