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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes Olga I. Guliy a
abc
a
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, Larisa Yu. Matora , Lev A. Dykman , Sergey A. Staroverov d
a
abc
, Gennady L.
a
Burygin , Viktor D. Bunin , Andrei M. Burov & Oleg V. Ignatov a
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian, Academy of Sciences (IBPPM RAS), 13 Prospekt Entuziastov, Saratov 410049, Russian Federation b
Vavilov Saratov State Agrarian University, 1 Teatralnaya Ploshchad, Saratov 410012, Russian Federation c
Saratov Veterinary Research Institute, 6 Ulitsa 53-ei Strelkovoi Divizii, Saratov 410028, Russian Federation d
Elosystems GmbH, Berlin, Germany Accepted author version posted online: 02 Oct 2014.
To cite this article: Olga I. Guliy, Larisa Yu. Matora, Lev A. Dykman, Sergey A. Staroverov, Gennady L. Burygin, Viktor D. Bunin, Andrei M. Burov & Oleg V. Ignatov (2014): Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes, Journal of Immunoassay and Immunochemistry, DOI: 10.1080/15321819.2014.955584 To link to this article: http://dx.doi.org/10.1080/15321819.2014.955584
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
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Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes
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Olga I. Guliy1;2;3, Larisa Yu. Matora1, Lev A. Dykman1;3, Sergey A. Staroverov1;2;3, Gennady L. Burygin1, Viktor D. Bunin4, Andrei M. Burov1, and Oleg V. Ignatov1
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1
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Sciences (IBPPM RAS), 13 Prospekt Entuziastov, Saratov 410049, Russian Federation
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Federation
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Short title: Exposure of Azospirillum brasilense carbohydrate epitopes
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
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Vavilov Saratov State Agrarian University, 1 Teatralnaya Ploshchad, Saratov 410012, Russian
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Russian Federation
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Paper subject category: Special topics
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Abbreviations: Ab, antibody; EO, electro-optical; LPS, lipopolysaccharide;
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OPS, O polysaccharide; PBS, phosphate-buffered saline
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*Correspondence and proofs:
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Olga I. Guliy
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russian Federation
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Tel.: +7(8452)970444. Fax: +7(8452)970383. E-mail: [email protected]
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Saratov Veterinary Research Institute, 6 Ulitsa 53-ei Strelkovoi Divizii, Saratov 410028,
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Elosystems GmbH, Berlin, Germany
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The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-
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optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO)
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properties of the suspensions, we used antibodies (Abs) generated to the complete
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lipopolysaccharide (LPS) of A. brasilense type strain Sp7 and also antibodies to the smooth and
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rough O polysaccharides of Sp7. After 18 h of culture growth, the EO signal of the suspension
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treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of
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the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 h of
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culture growth, the strongest EO signal was observed for the cells treated with antibodies to
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rough O polysaccharide (approximately 46% greater than the control), whereas for the cells
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treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of
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the control). These data were confirmed by electron microscopy. The results of the study may
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have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial
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biotechnology, when the antigenic composition of the bacterial surface requires close control.
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Keywords:
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Azospirillum brasilense;
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Antigen carbohydrate epitopes;
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Electrophysical properties;
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Antibodies;
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Determination of microorganisms
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ABSTRACT
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Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative
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bacteria. It is not an individual chemical compound but represents a family of molecules that
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differ in structure and molecular weight [1]. The complete LPS macromolecule consists of a
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polysaccharide portion (O-specific chain, also known as O antigen or OPS) and a core
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oligosaccharide, which is covalently attached to a lipid component termed lipid A [2]. In LPSs,
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certain antigenic determinants can be exposed at some phases of culture growth and shielded at
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other phases. For example, by immunofluorescence and enzyme-linked immunosorbent assays,
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new antigenic determinants of LPS were found to appear at the late lag-phase and at the early
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stationary phase of Rhizobium trifolii strain 0403 [3]. These determinants were not detectable
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either in the mid-exponential phase or at the end of the stationary phase of culture growth.
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The LPSs of Azospirillum bacteria are immunochemically heterogeneousone LPS has several
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O polysaccharides (OPSs) as part of its structure, which differ in their antigenic determinants
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[4;5]. In particular, A. brasilense type strain Sp7 has two OPSsa rough one (R-OPS) and a
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smooth one (S-OPS). These designations were introduced by Matora et al. [5] to show that it is
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the redistribution of contributions of these antigenic structures to the structure of the cell surface
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INTRODUCTION
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that determines the nature of the atypical R-to-S dissociation in this strain. It was found that after
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18 h of cultivation of A. brasilense wild R-type strain Sp7, both OPSs were immunogenic and
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that further cultivation led to predominant synthesis of R-OPS. This, together with the shielding
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effect toward the second OPS, ensures the characteristic colony morphology and the loss of
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immunogenicity by S-OPS after 72 h of cell cultivation. 3
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result in the predominance of specific antigenic determinants, which should be considered when
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appropriate antibodies for the detection of bacteria are being selected.
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In the past 15 years, the electrophysical methods of electrorotation and cell electro-orientation in
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an AC field have enjoyed active use for assessing various physiological parameters of bacterial
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cells [6–7]. We have shown previously that the electro-optical (EO) analysis of cell suspensions
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is applicable to the detection of cells with the help of mono- and polyclonal antibodies (Abs) [8–
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9].
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An important element in creating an EO method for immunological detection of cells is the
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selection of antibodies that would ensure high sensitivity of the method. We assumed that the
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interaction of bacterial cells with Abs to R- and S-antigen at different stages of culture growth
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would lead to different changes in the magnitude of the EO signal.
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The aim of this work was to determine changes in the antigenic properties of the LPS of
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microbial cells during culture growth, by using EO analysis of cell suspensions.
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MATERIALS AND METHODS
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Thus, changes in the antigenic structure of the microbial surface during cell cultivation will
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Strains and culture conditions
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A. brasilense Sp7, Sp245, Sp7.K.2, and Cd were obtained from the culture collection held at the
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Russian Academy of Sciences’ Institute of Biochemistry and Physiology of Plants and
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Microorganisms (Saratov, Russia). Bacteria were grown in Döbereiner and Day’s [10] liquid 4
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± 1ºC for 24 h.
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Microbial cells were stored at + 4°C in a Petri dish with a potato agar (3%) and were subcultured
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every 2 weeks.
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Antibodies
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Abs to cells of all four A. brasilense strains were used. For immunization with intact cells of A.
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brasilense Sp7, we used cell suspensions (absorbance A660 of 0.5, cell concentration of ~109 cells
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ml-1) in a 1-cm-thick cuvette. At four weekly intervals, rabbits received multiple intracutaneous
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injections of 1 ml of a suspension mixed with an equal volume of Freund’s adjuvant. Abs against
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the LPSs of A. brasilense Sp7, Cd, and mutant strain Sp7.K.2 were raised as described by Matora
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et al. [11]. The immunoglobulin G (IgG) fractions were obtained by antiserum fractionation with
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40% ammonium sulfate followed by dialysis against phosphate-buffered saline (PBS) and by
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affinity chromatography on a column of protein A–Sepharose 4B (Sigma, USA). The Abs were
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diluted with PBS to the desired working concentration. The IgG concentration in the solutions
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was determined spectrophotometrically at 278 nm, by assuming the absorbance of the IgG
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solution in the cuvette (protein concentration of 1 mg ml-1) to be 1.4 [12].
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synthetic malate medium supplemented with NH4CL (1 g l-1) on a rotary shaker (120 rpm) at 28
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In simultaneous control experiments to study the interaction of the Sp7 cells with nonspecific
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Abs, we used Abs against the O antigen of A. brasilense Sp245. These were raised as described
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by Matora et al. [11].
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Before analysis, the cells were washed three times with distilled water by (conductivity of 1.8 µS
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cm-1) centrifugation at 2800 × g for 5 min and then were resuspended in a little water sufficient
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for re-suspension. To remove cellular aggregates, we centrifuged the cell suspension again at 110
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× g for 1 min and used the suspension that remained in the supernatant liquid for further work.
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The A660 of the suspension prepared in this way was adjusted to 0.4–0.42.
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A. brasilense interaction with Abs
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Abs were added to A. brasilense suspensions (prepared as described above) to a final
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concentration of 1.0 mg ml-1, and the suspensions were incubated at 37°C for 30 min.
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Suspensions incubated without Abs were used as controls. After incubation, the cells were
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washed three times with distilled water (1.6–2.0 µS cm-1) and were used for EO measurements.
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EO analysis
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The orientational spectra of the cells were measured as described previously [13], with the help
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of the ELUS EO analyzer, developed by the State Research Center for Applied Microbiology,
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Obolensk, Russia. A discrete set of frequencies of the orienting electric field (10, 100, 250, and
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500 kHz) was used. Five independent experiments for each point were made, and Excel 2003
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software was used to calculate error bars.
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Preparation of cells for EO analysis
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For electron microscopic identification of the A. brasilense Sp7–Ab interaction, preparations
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containing bacterial cells at 106 cells ml-1 were made. A 0.5-ml portion of a bacterial suspension
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in PBS was incubated with 10 µl of homologous Abs (concentration of 100 µg ml-1) on a shaker
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for 1 h. Then, the suspension was centrifuged at 12000 × g for 5 min, and the cell precipitate was
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resuspended in 0.5 ml of PBS. The suspension was incubated with 100 µl of protein A–colloidal
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gold (A520 = 0.5; IBPPM RAS, Russia) for 1 h. After repeated sedimentation and resuspending,
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approximately 20 µl of the suspension was placed on parafilm and a 200-mesh nickel grid
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(Balzers, Liechtenstein) with carbon-reinforced nitrocellulose film was placed on top of the drop
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for 20 min. Heat fixing was performed by keeping the grid near an incandescent lamp for 2 min.
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Excess liquid was removed by touching a filter paper strip with the grid. The grid then was
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rinsed by transfer onto a drop of triply distilled water, dried, and viewed with a Libra 120
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electron microscope (Carl Zeiss, Germany) at an accelerating voltage of 120 kV.
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RESULTS
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In this study, we used A. brasilense type strain Sp7, whose LPS (a major carbohydrate antigen)
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Electron microscopy
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contains two OPSs. Matveev et al. [14] were the first to describe an R-to-S dissociation in A.
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brasilense Sp7.The OPSs of Sp7 had previously been designated by us as R-OPS and S-OPS, as
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the redistribution of contributions from these antigenic structures to cell surface composition
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determines the character of R-to-S dissociation in this strain [15]. Changes in the electrophysical
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properties of the cell suspensions were studied with Abs generated to the complete LPS of Sp7 7
and with Abs to each of the OPS. To raise Abs toward R-OPS, we used a derivative of strain
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Sp7, A. brasilense Sp7.K.2, which has only R-OPS as part of its LPS [16]. To detect S-OPS, we
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used Abs to the LPS of a closely related strain, Cd, which recognize only the S-OPS of Sp7 [17].
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The OPS changes during cell life were evaluated by EO analysis and by electron microscopy.
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For EO analysis, the cells were grown in a mineral medium, and the EO properties of bacterial
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suspensions were studied during the interaction of the cells with Abs to the various determinants.
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Fig. 1 shows electro-orientation spectra of A. brasilense Sp7 grown for 18 h (A) and 72 h (С) in
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a liquid mineral medium. The spectra were taken after cell interaction with anti-LPS Abs. The
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spectra changed across the range of the frequencies used740, 1000, 1450, 2000, and 2800 kHz.
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After 18 h of culture growth, the EO signal of the suspension treated with anti-S-OPS Abs was
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approximately 20% lower than that of the suspension treated with anti-LPS Abs (control),
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whereas the EO signal for the suspension treated with anti-R-OPS Abs was approximately 25%
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of the control (Fig. 1B).
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The electron microscopic results (Fig. 2A) confirmed that anti-LPS Abs interacted at a high level
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with Sp7 cells. After culture growth for 18 h, anti-S-OPS Abs interacted with Sp7 cells, as
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detected by the colloidal gold label (Fig. 2B). Anti-R-OPS Abs interacted at a high level with
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Sp7 cells after growth for 72 h (Fig. 2C).
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After 72 h of culture growth (Fig. 1C and D), the strongest EO signal was observed for the cells
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treated with anti-R-OPS Abs (approximately 46% greater than the control), whereas for the cells
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treated with anti-S-OPS Abs, it was much lower (approximately 23% of the control; Fig. 2D).
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These results indicate that during culture growth, R-OPS shields the other OPS, and the 8
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for the morphological peculiarities of the R colonies of A. brasilense Sp7. Thus, the EO data are
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in good agreement with the results of immunoelectron microscopy (Fig. 2).
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In simultaneous control experiments to study the interaction of the Sp7 cells with nonspecific
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Abs, we used Abs raised against the O antigen of A. brasilense Sp245. No changes in the EO
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signal magnitude were recorded.
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DISCUSSION
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The fact that certain antigenic determinants of LPS can be exposed at some phases of culture
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growth and shielded at other phases is important for microbiological diagnostics, particularly for
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the Ab-aided detection of microbial cells.
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Because the EO results accord well with the electron microscopic data, we believe that the
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method put forward in this paper allows effective assessment of the degree of epitope exposure
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on the bacterial-cell surface during culture growth. Furthermore, the obtained results can serve as
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a basis for the development of a rapid test for the interspecies detection of bacterial R- or S-
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forms.
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preferential synthesis of R-OPS at the relatively late stages of culture growth seems responsible
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EO analysis takes much less time than do other techniques because of a decreased period of
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sample preparation. In addition, EO analysis allows sample preparation to be fully automated
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and a large number of experimental samples to be analyzed [18].
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We conclude that the EO analysis of bacterial suspensions can be used to determine changes in
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the exposure of the carbohydrate epitopes of bacterial outer membranes. The results of the study
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may have importance for the rapid evaluation of changes in LPS form in microbial
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biotechnology, when the antigenic composition of the bacterial surface requires close control.
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We thank D.N. Tychinin (IBPPM RAS, Saratov, Russia) for technical assistance.
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Figure 1. EO signals for suspensions of A. brasilense Sp7 grown for 18 (A) and 72 h (C) after cell interaction with various Abs. 1, Ab-free control. 2, O-specific Abs. 3, Anti-S-OPS Abs. 4, Anti-R-OPS Abs. (B, D) Changes in the relative unit values at an orienting field frequency of 1000 kHz (control minus experiment).
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Figure 2. Electron micrograph of A. brasilense Sp7 labeled with protein A–colloidal gold after cell interaction with strain-specific Abs to the complete LPS of Sp7 (A), Abs to A. brasilense Cd (B), and Abs to A. brasilense Sp7.K.2 (C).
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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes Olga I. Guliy a
abc
a
ac
, Larisa Yu. Matora , Lev A. Dykman , Sergey A. Staroverov d
a
abc
, Gennady L.
a
Burygin , Viktor D. Bunin , Andrei M. Burov & Oleg V. Ignatov a
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian, Academy of Sciences (IBPPM RAS), 13 Prospekt Entuziastov, Saratov 410049, Russian Federation b
Vavilov Saratov State Agrarian University, 1 Teatralnaya Ploshchad, Saratov 410012, Russian Federation c
Saratov Veterinary Research Institute, 6 Ulitsa 53-ei Strelkovoi Divizii, Saratov 410028, Russian Federation d
Elosystems GmbH, Berlin, Germany Accepted author version posted online: 02 Oct 2014.
To cite this article: Olga I. Guliy, Larisa Yu. Matora, Lev A. Dykman, Sergey A. Staroverov, Gennady L. Burygin, Viktor D. Bunin, Andrei M. Burov & Oleg V. Ignatov (2014): Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes, Journal of Immunoassay and Immunochemistry, DOI: 10.1080/15321819.2014.955584 To link to this article: http://dx.doi.org/10.1080/15321819.2014.955584
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
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Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes
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Olga I. Guliy1;2;3, Larisa Yu. Matora1, Lev A. Dykman1;3, Sergey A. Staroverov1;2;3, Gennady L. Burygin1, Viktor D. Bunin4, Andrei M. Burov1, and Oleg V. Ignatov1
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Sciences (IBPPM RAS), 13 Prospekt Entuziastov, Saratov 410049, Russian Federation
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Federation
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Short title: Exposure of Azospirillum brasilense carbohydrate epitopes
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
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Vavilov Saratov State Agrarian University, 1 Teatralnaya Ploshchad, Saratov 410012, Russian
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Russian Federation
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Paper subject category: Special topics
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Abbreviations: Ab, antibody; EO, electro-optical; LPS, lipopolysaccharide;
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OPS, O polysaccharide; PBS, phosphate-buffered saline
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*Correspondence and proofs:
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Olga I. Guliy
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russian Federation
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Tel.: +7(8452)970444. Fax: +7(8452)970383. E-mail: [email protected]
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Saratov Veterinary Research Institute, 6 Ulitsa 53-ei Strelkovoi Divizii, Saratov 410028,
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Elosystems GmbH, Berlin, Germany
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The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-
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optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO)
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properties of the suspensions, we used antibodies (Abs) generated to the complete
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lipopolysaccharide (LPS) of A. brasilense type strain Sp7 and also antibodies to the smooth and
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rough O polysaccharides of Sp7. After 18 h of culture growth, the EO signal of the suspension
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treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of
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the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 h of
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culture growth, the strongest EO signal was observed for the cells treated with antibodies to
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rough O polysaccharide (approximately 46% greater than the control), whereas for the cells
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treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of
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the control). These data were confirmed by electron microscopy. The results of the study may
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have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial
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biotechnology, when the antigenic composition of the bacterial surface requires close control.
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Keywords:
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Azospirillum brasilense;
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Antigen carbohydrate epitopes;
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Electrophysical properties;
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Antibodies;
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Determination of microorganisms
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ABSTRACT
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Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative
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bacteria. It is not an individual chemical compound but represents a family of molecules that
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differ in structure and molecular weight [1]. The complete LPS macromolecule consists of a
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polysaccharide portion (O-specific chain, also known as O antigen or OPS) and a core
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oligosaccharide, which is covalently attached to a lipid component termed lipid A [2]. In LPSs,
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certain antigenic determinants can be exposed at some phases of culture growth and shielded at
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other phases. For example, by immunofluorescence and enzyme-linked immunosorbent assays,
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new antigenic determinants of LPS were found to appear at the late lag-phase and at the early
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stationary phase of Rhizobium trifolii strain 0403 [3]. These determinants were not detectable
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either in the mid-exponential phase or at the end of the stationary phase of culture growth.
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The LPSs of Azospirillum bacteria are immunochemically heterogeneousone LPS has several
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O polysaccharides (OPSs) as part of its structure, which differ in their antigenic determinants
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[4;5]. In particular, A. brasilense type strain Sp7 has two OPSsa rough one (R-OPS) and a
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smooth one (S-OPS). These designations were introduced by Matora et al. [5] to show that it is
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the redistribution of contributions of these antigenic structures to the structure of the cell surface
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INTRODUCTION
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that determines the nature of the atypical R-to-S dissociation in this strain. It was found that after
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18 h of cultivation of A. brasilense wild R-type strain Sp7, both OPSs were immunogenic and
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that further cultivation led to predominant synthesis of R-OPS. This, together with the shielding
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effect toward the second OPS, ensures the characteristic colony morphology and the loss of
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immunogenicity by S-OPS after 72 h of cell cultivation. 3
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result in the predominance of specific antigenic determinants, which should be considered when
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appropriate antibodies for the detection of bacteria are being selected.
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In the past 15 years, the electrophysical methods of electrorotation and cell electro-orientation in
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an AC field have enjoyed active use for assessing various physiological parameters of bacterial
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cells [6–7]. We have shown previously that the electro-optical (EO) analysis of cell suspensions
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is applicable to the detection of cells with the help of mono- and polyclonal antibodies (Abs) [8–
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9].
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An important element in creating an EO method for immunological detection of cells is the
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selection of antibodies that would ensure high sensitivity of the method. We assumed that the
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interaction of bacterial cells with Abs to R- and S-antigen at different stages of culture growth
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would lead to different changes in the magnitude of the EO signal.
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The aim of this work was to determine changes in the antigenic properties of the LPS of
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microbial cells during culture growth, by using EO analysis of cell suspensions.
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MATERIALS AND METHODS
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Thus, changes in the antigenic structure of the microbial surface during cell cultivation will
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Strains and culture conditions
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A. brasilense Sp7, Sp245, Sp7.K.2, and Cd were obtained from the culture collection held at the
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Russian Academy of Sciences’ Institute of Biochemistry and Physiology of Plants and
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Microorganisms (Saratov, Russia). Bacteria were grown in Döbereiner and Day’s [10] liquid 4
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± 1ºC for 24 h.
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Microbial cells were stored at + 4°C in a Petri dish with a potato agar (3%) and were subcultured
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every 2 weeks.
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Antibodies
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Abs to cells of all four A. brasilense strains were used. For immunization with intact cells of A.
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brasilense Sp7, we used cell suspensions (absorbance A660 of 0.5, cell concentration of ~109 cells
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ml-1) in a 1-cm-thick cuvette. At four weekly intervals, rabbits received multiple intracutaneous
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injections of 1 ml of a suspension mixed with an equal volume of Freund’s adjuvant. Abs against
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the LPSs of A. brasilense Sp7, Cd, and mutant strain Sp7.K.2 were raised as described by Matora
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et al. [11]. The immunoglobulin G (IgG) fractions were obtained by antiserum fractionation with
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40% ammonium sulfate followed by dialysis against phosphate-buffered saline (PBS) and by
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affinity chromatography on a column of protein A–Sepharose 4B (Sigma, USA). The Abs were
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diluted with PBS to the desired working concentration. The IgG concentration in the solutions
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was determined spectrophotometrically at 278 nm, by assuming the absorbance of the IgG
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solution in the cuvette (protein concentration of 1 mg ml-1) to be 1.4 [12].
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synthetic malate medium supplemented with NH4CL (1 g l-1) on a rotary shaker (120 rpm) at 28
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In simultaneous control experiments to study the interaction of the Sp7 cells with nonspecific
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Abs, we used Abs against the O antigen of A. brasilense Sp245. These were raised as described
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by Matora et al. [11].
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Before analysis, the cells were washed three times with distilled water by (conductivity of 1.8 µS
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cm-1) centrifugation at 2800 × g for 5 min and then were resuspended in a little water sufficient
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for re-suspension. To remove cellular aggregates, we centrifuged the cell suspension again at 110
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× g for 1 min and used the suspension that remained in the supernatant liquid for further work.
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The A660 of the suspension prepared in this way was adjusted to 0.4–0.42.
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A. brasilense interaction with Abs
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Abs were added to A. brasilense suspensions (prepared as described above) to a final
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concentration of 1.0 mg ml-1, and the suspensions were incubated at 37°C for 30 min.
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Suspensions incubated without Abs were used as controls. After incubation, the cells were
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washed three times with distilled water (1.6–2.0 µS cm-1) and were used for EO measurements.
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EO analysis
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The orientational spectra of the cells were measured as described previously [13], with the help
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of the ELUS EO analyzer, developed by the State Research Center for Applied Microbiology,
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Obolensk, Russia. A discrete set of frequencies of the orienting electric field (10, 100, 250, and
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500 kHz) was used. Five independent experiments for each point were made, and Excel 2003
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software was used to calculate error bars.
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Preparation of cells for EO analysis
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For electron microscopic identification of the A. brasilense Sp7–Ab interaction, preparations
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containing bacterial cells at 106 cells ml-1 were made. A 0.5-ml portion of a bacterial suspension
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in PBS was incubated with 10 µl of homologous Abs (concentration of 100 µg ml-1) on a shaker
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for 1 h. Then, the suspension was centrifuged at 12000 × g for 5 min, and the cell precipitate was
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resuspended in 0.5 ml of PBS. The suspension was incubated with 100 µl of protein A–colloidal
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gold (A520 = 0.5; IBPPM RAS, Russia) for 1 h. After repeated sedimentation and resuspending,
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approximately 20 µl of the suspension was placed on parafilm and a 200-mesh nickel grid
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(Balzers, Liechtenstein) with carbon-reinforced nitrocellulose film was placed on top of the drop
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for 20 min. Heat fixing was performed by keeping the grid near an incandescent lamp for 2 min.
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Excess liquid was removed by touching a filter paper strip with the grid. The grid then was
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rinsed by transfer onto a drop of triply distilled water, dried, and viewed with a Libra 120
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electron microscope (Carl Zeiss, Germany) at an accelerating voltage of 120 kV.
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RESULTS
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In this study, we used A. brasilense type strain Sp7, whose LPS (a major carbohydrate antigen)
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Electron microscopy
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contains two OPSs. Matveev et al. [14] were the first to describe an R-to-S dissociation in A.
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brasilense Sp7.The OPSs of Sp7 had previously been designated by us as R-OPS and S-OPS, as
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the redistribution of contributions from these antigenic structures to cell surface composition
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determines the character of R-to-S dissociation in this strain [15]. Changes in the electrophysical
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properties of the cell suspensions were studied with Abs generated to the complete LPS of Sp7 7
and with Abs to each of the OPS. To raise Abs toward R-OPS, we used a derivative of strain
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Sp7, A. brasilense Sp7.K.2, which has only R-OPS as part of its LPS [16]. To detect S-OPS, we
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used Abs to the LPS of a closely related strain, Cd, which recognize only the S-OPS of Sp7 [17].
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The OPS changes during cell life were evaluated by EO analysis and by electron microscopy.
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For EO analysis, the cells were grown in a mineral medium, and the EO properties of bacterial
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suspensions were studied during the interaction of the cells with Abs to the various determinants.
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Fig. 1 shows electro-orientation spectra of A. brasilense Sp7 grown for 18 h (A) and 72 h (С) in
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a liquid mineral medium. The spectra were taken after cell interaction with anti-LPS Abs. The
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spectra changed across the range of the frequencies used740, 1000, 1450, 2000, and 2800 kHz.
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After 18 h of culture growth, the EO signal of the suspension treated with anti-S-OPS Abs was
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approximately 20% lower than that of the suspension treated with anti-LPS Abs (control),
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whereas the EO signal for the suspension treated with anti-R-OPS Abs was approximately 25%
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of the control (Fig. 1B).
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The electron microscopic results (Fig. 2A) confirmed that anti-LPS Abs interacted at a high level
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with Sp7 cells. After culture growth for 18 h, anti-S-OPS Abs interacted with Sp7 cells, as
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detected by the colloidal gold label (Fig. 2B). Anti-R-OPS Abs interacted at a high level with
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Sp7 cells after growth for 72 h (Fig. 2C).
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After 72 h of culture growth (Fig. 1C and D), the strongest EO signal was observed for the cells
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treated with anti-R-OPS Abs (approximately 46% greater than the control), whereas for the cells
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treated with anti-S-OPS Abs, it was much lower (approximately 23% of the control; Fig. 2D).
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These results indicate that during culture growth, R-OPS shields the other OPS, and the 8
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for the morphological peculiarities of the R colonies of A. brasilense Sp7. Thus, the EO data are
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in good agreement with the results of immunoelectron microscopy (Fig. 2).
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In simultaneous control experiments to study the interaction of the Sp7 cells with nonspecific
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Abs, we used Abs raised against the O antigen of A. brasilense Sp245. No changes in the EO
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signal magnitude were recorded.
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DISCUSSION
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The fact that certain antigenic determinants of LPS can be exposed at some phases of culture
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growth and shielded at other phases is important for microbiological diagnostics, particularly for
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the Ab-aided detection of microbial cells.
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Because the EO results accord well with the electron microscopic data, we believe that the
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method put forward in this paper allows effective assessment of the degree of epitope exposure
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on the bacterial-cell surface during culture growth. Furthermore, the obtained results can serve as
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a basis for the development of a rapid test for the interspecies detection of bacterial R- or S-
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forms.
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preferential synthesis of R-OPS at the relatively late stages of culture growth seems responsible
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EO analysis takes much less time than do other techniques because of a decreased period of
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sample preparation. In addition, EO analysis allows sample preparation to be fully automated
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and a large number of experimental samples to be analyzed [18].
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We conclude that the EO analysis of bacterial suspensions can be used to determine changes in
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the exposure of the carbohydrate epitopes of bacterial outer membranes. The results of the study
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may have importance for the rapid evaluation of changes in LPS form in microbial
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biotechnology, when the antigenic composition of the bacterial surface requires close control.
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We thank D.N. Tychinin (IBPPM RAS, Saratov, Russia) for technical assistance.
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Figure 1. EO signals for suspensions of A. brasilense Sp7 grown for 18 (A) and 72 h (C) after cell interaction with various Abs. 1, Ab-free control. 2, O-specific Abs. 3, Anti-S-OPS Abs. 4, Anti-R-OPS Abs. (B, D) Changes in the relative unit values at an orienting field frequency of 1000 kHz (control minus experiment).
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Figure 2. Electron micrograph of A. brasilense Sp7 labeled with protein A–colloidal gold after cell interaction with strain-specific Abs to the complete LPS of Sp7 (A), Abs to A. brasilense Cd (B), and Abs to A. brasilense Sp7.K.2 (C).
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