Biomedical Chemistry: Research and Methods 2021, 4(4), e00154

Development of a method for the extraction of the total proteome of bacillus anthracis spores

E.A. Koteneva1,2*, O.I. Tsygankova1, A.V. Kalinin1, V.Yu. Shcherbakova1, I.S. Rodionov1, V.V. Serdyukov1, A.V. Abramovich1

1Stavropol Research Anti-Plague Institute, 13-15, Sovetskaya str., Stavropol, 355035 Russia
2North Caucasus Federal University, Stavropol, 355009 Russia;*e-mail: postgenom_stv@mail.ru

Keywords:Bacillus anthracis; spore proteome; extraction of the proteomic complex; lysis solutions; disintegration; 2D-gel electrophoresis

DOI:10.18097/BMCRM00154

The whole version of this paper is available in Russian.

We have developed a method for obtaining the total proteome of Bacillus anthracis spores, which combines efficient protein extraction with reliable disinfection of samples. We used 7 strains of B. anthracis: 4 plasmid-free, 3 diplasmid, one of which with an atypical type of capsule formation in air. The schemes for isolating the total spore proteome were tested using various lysis solutions in the presence of bacterial protease inhibitors, with the inclusion of the stages of treatment of spores with trichloroacetic acid and mechanical disintegration and with their exclusion. The quality and completeness of the extraction of the total proteome of the spores of the samples was assessed in one-dimensional (1D) and two- dimensional (2D) electrophoresis. Treatment of spores with trichloroacetic acid increased the reliability of material disinfection and reduces the loss of the final product. Mechanical disintegration after treatment of spores with lysing solutions increases the completeness of extraction of spore proteins in a wide range of molecular weights and facilitated the process of sterilizing filtration. Final filtration of the lysate through a PVDF filter with a pore size of 0.22 µm provided additional decontamination of samples without reducing their quality. Thus, the use of the proposed sample preparation scheme makes it possible to obtain complete protein extracts of spores of B. anthracis strains, suitable for comparative analysis of the proteome and search for a possible correlation with the features phenotypic properties and mechanisms that ensure the preservation of the pathogen anthrax in environmental objects, including soil
Figure 1. Research scheme for the development of an extraction algorithm for the proteomic complex of B. anthracis spores.

Figure 2. The dynamics of changes in the appearance of the spores of the B. antracis 1 (CO) -23 strain in the process of obtaining the proteomic complex:
a - after rinsing and washing; b - after treatment with TCA; c - after treatment with lysis buffer with urea; d - after additional mechanical disintegration; e - after treatment with a lysis solution with guanidine chloride; f - after additional mechanical disintegration. Rebiger staining. Magnification 100x5.

Figure 3. Electrophoregram of protein extracts obtained with and without mechanical disintegration.
L- Experion Pro260 ladder calibration scale. The calibration scale contains 9 proteins with a mass range from 10 to 260 kDa, as well as a 1.2 kDa marker. The electrophoretogram of the calibration scale displays a lower 1.2 kDa marker following the system peaks and 9 proteins on the scale. Protein peaks in each sample well are capped at 1.2 kDa for the lower marker and 260 kDa for the upper marker. These markers are used to normalize the time of protein migration into sample wells using a calibration bar. (There is always a system peak after the 1.2 kDa low marker.)
1-4 protein extracts obtained using mechanical disintegration, 5-8 protein extracts obtained without the use of mechanical disintegration. 1, 5 - strain B. anthracis ∆Sterne in lysis buffer with urea, 2, 6- strain B. anthracis ∆Sterne in lysis buffer with guanidine chloride, 3, 7 - strain B. anthracis 1284 in lysis buffer with urea, 4, 8 - B. anthracis strain 1284 in lysis buffer with guanidine chloride.

Figure 4. 2D electrophoretogram of the total spore proteome extracts of the B. anthracis 1 (CO) -23 strain, zinc staining. Strip pH 4-7.

CLOSE
Table 1. Lysis buffers used for total proteome extraction from B. anthracis spore suspension.

FUNDING

The study was carried out on the topic of research work as part of the implementation of the Rospotrebnadzor Sectoral Research Program for the period 2021-2025. “Scientifc support of epidemiological surveillance and sanitary protection of the territory of the Russian Federation. Creation of new technologies, means and methods of control and prevention of infectious and parasitic diseases”.

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