The Role of Vesicles in Transporting of Cholera Toxin

The review reports on the secretion pathways of the main virulence factor of Vibrio cholerae, cholera toxin, both through the two-stage Sec-dependent type 2 secretion system and with the help of vesicles of the outer membrane of V. cholerae. The ways of toxin transfer into the host organism, depending on its form, are discussed. The well-studied free soluble cholera toxin is secreted extracellularly and transmitted in a GM1-dependent manner through cholesterolrich lipid rafts. The transfer of cholera toxin associated with vesicles has advantages over free toxin, because substances inside the outer membrane vesicles are protected from external proteases and host antibodies by the membrane that forms the vesicle. Vesicular transporting of cholera toxin into the target cell occurs via clathrin-dependent, caveolin-dependent and lipid raft-dependent endocytosis. The specific transport route is determined by the structure of the vesicles. Clathrindependent endocytosis is described for V. cholerae strains cultivated at low osmolarity of the medium, whose outer membrane vesicles contain the cholera toxin subunit A inside. Lipid raft-dependent endocytosis is characteristic of vesicles in which cholera toxin is located on the surface. In addition, endocytosis of V. cholerae outer membrane vesicles through structures known as caveolae is presented

THE STUDY OF DIAGNOSTIC POTENTIAL OF MONOCLONAL ANTIBODIES SPECIFIC TO THE MEMBRANE PROTEIN OF CHOLERA AGENT IN ENZYME-LINKED IMMUNOASSAY

Objective of the study was to develop peroxidase conjugate on the base of monoclonal antibodies (MCA) H2F6 and to study the possibility of its application for the detection of tcp+ Vibrio cholerae O1/O139 strains using direct ELISA methods.Materials and methods. Utilized for the investigation was the hybrid H2F6 clone, which synthesized monoclonal antibodies specific to the outer membrane protein of cholera vibrio into culture medium.Results and conclusions. Peroxidase conjugate was designed on the base of MCA which allows for the detection of tcp+ V. cholerae O1 and O139 strains in direct solid-phase enzyme-linked immunosorbent assay and dot-ELISA. The preparation was tested on a group of strains of V. cholerae and heterologous microorganisms and showed specificity in relation to V. cholerae O1 and O139. Monoclonal peroxidase conjugate H2F6 can be used for the detection of epidemically significant V. cholerae O1/O139 strains by means of immune-enzyme methods

THE ROLE OF YERSINIA PESTIS RESIDENT PLASMIDS PMT1, PCD1, AND PPCP1 IN THE PRODUCTION OF LIPOPOLYSACCHARIDE EXTRACELLULAR FORM

Objective of the study is to investigate the role of resident plasmids pMT1, pCD1, and pPCP1 in the production of extracellular form of Yersinia pestis lipopolysaccharide (LPS).Materials and methods. The experiments have been performed using Y. pestis strain EV76 (pMT1, pCD1, pPCP1), carrying the whole plasmid set, as well as plasmid-free Y. pestis variant EV76 (pMT1-, pCD1-, pPCP1-), and isogenic clones, harbouring only one plasmid: Y. pestis EV76 (pMT1); Y. pestis EV76 (pCD1); Y. pestis EV76 (pPCP1). The presence of extracellular LPS in the incubation medium of Y. pestis EV76 cells has been confirmed by supernatant toxicity for laboratory animals and also by LAL-test reaction.Results and conclusions. It has been established that LPS extracellular form is produced by 37 °C Y. pestis EV76 cultures of the initial strain and its variants, carrying pMT1 or pPCP1 plasmid. Plasmid-free cultures and variant harbouring pCPP1 plasmid are deprived of such ability. The results of LAL-test has shown that the process of LPS separation from cell wall membrane into the environment is associated with translocation of proteins encoded by pMT1 and pCD1 plasmids and constitutes a natural form of existence of Y. pestis cells. The involvement of pCD1 plasmid in realization of the toxic potential of Y. pestis LPS has been established for the first time ever

Study of the Surface Antigenic Determinants of <i>Vibrio cholerae</i> Strains with Atypical Agglutinability Using the Panel of Monoclonal Antibodies

The aim of the work was to study surface antigenic determinants of V. cholerae R-variant strains using enzyme immunoassay and a panel of monoclonal antibodies (MAbs).Materials and methods. 60 strains of V. cholerae R-variant isolated from ambient environment objects in the territories of the former USSR and the constituent entities of the Russian Federation over a 30-year period (1988–2019) were investigated in the slide agglutination reaction with cholera diagnostic sera, enzyme immunoassay (ELISA) using the panel of MAbs specific to membrane proteins and a set of reagents “Monoclonal diagnostic immunoglobulins labeled with horseradish peroxidase, dry, for serological identification of V. cholerae O1 and O139 (in vitro) through ELISA and dot-ELISA”.Results and discussion. The analysis of the surface structures of V. cholerae R-variant strains with atypical agglutinability has been carried out applying enzyme immunoassay. It showed that individual strains with different amounts of O-antigen are registered among the studied strains identified at isolation as V. cholerae R-variant (the optical density range is from 0.261±0.002 to 1.312±0.003). Epitopes of specific O-antigen were found in some “conservative” strains (30 %) that are agglutinated only with RO serum, and in several strains (20 %) that do not have the wbeT gene that determines its synthesis, and lost agglutinability with all diagnostic cholera sera, including RO. The protein epitopes recognized by complementary MAbs are represented with varying frequency in the composition of surface antigens of R-vibrios; a decrease in their representation or absence on the cell surface correlates with the modification or loss of R-LPS and is accompanied by a negative agglutination reaction

Yersinia pestis pathogenicity

Yersinia pestis belongs to those pathogenic bacteria which produce lipopolysaccharide (LPS) having the function of a toxin. In order to make a toxic effect the polymer must be separated from the cell outer membrane and presented to the immunocompetent cell receptors of the host in the functionally active form. In this review data of russian and foreign investigators on Y. pestis toxigenic properties was presented. Results of the authors' own experiments showing that Y. pestis is able to export LPS into the surrounding medium are included. This process is a natural function of the living cell, is realized at 37 degrees C and is strictly dependent on the expression of Y. pestis genes of extrachromosomal inheritance, pMT1, pCD1, pPCPl. By the use of isogenic variants of Y. pestis EV76 vaccine strain and virulent 231 strain containing different plasmid combinations, it was established that maximum contribution in the activation of «high-temperature» LPS and its transformation into extracellular form made the proteins encoded by pCD1. The significance of the «murine» toxin encoded by pMT1 plasmid was less pronounced. The participation of pPCPl plasmid in the toxic effect was not discovered. The role of Y. pestis capsular substance and the significance of biologically active factors in the realization of Y. pestis LPS toxic potential is discussed. Functional relationship between translocation of the proteins encoded by plasmids and Y. pestis toxigenicity suggests Y. pestis biological uniqueness

SPECIFICITY OF IMMUNE MODULATING EFFECT OF YERSINIA PESTIS ENDOTOXIN

Literature and own data on mechanisms of realization of lipopolysaccharide (LPS) toxic potential of Yersinia pestis in the conditions of a macroorganism are analyzed. 2 modifications of LPS are examined - temperature dependent changes of chemical structure of polymers and a change in their conformation under the effect of micro- and macroorganism factors. A special attention is paid to comparative study of toxic and immune modulating properties of the specified LPS forms. Both LPS forms are concluded to activate TLR4/MD2 receptor, inducing synthesis of 2 types of cytokines - pro-inflammatory and interferons. However, dominance of their signal pathways and cross-regulation of the transduced signal are mirrored, and as a result the initial form of LPS initiates interferon synthesis, and conformationally changed - pro-inflammatory cytokines. Results of the experiments are summarized in 2 schemes of signal transfer by TLR4/MD2 receptor under the effect of 2 forms of Y. pestis LPS. Variations of cytokine-inducing properties of the initial and conformationally-altered forms of Y. pestis LPS corresponds to the immune response of the organism at each stage of the infectious process: late inflammatory response by interferon type is characteristic for intra-cellular cycle of plague development, and pro-inflammatory cytokine hyper-production is observed at the terminal stage of infection-toxic shock

EFFECT OF EXTRACHROMOSOMAL ELEMENTS OF HEREDITY ON TOXIC PROPERTIES OF YERSINIA PESTIS

Aim. Elucidation of the role of extrachromosomal elements of heredity in manifestations of toxic properties of Yersinia pestis. Materials and methods. The study was carried out in vaccine strain Y. pestis EV76 (pMTl, pCDl, pPCPl) and non-plasmid variants of vaccine EV76 (pMTl\ pCDl', pPCPl') and virulent 231 (рМТГ, pCDl’, pPCPl') strains of Y. pestis. Presence of functionally active form of lipopolysaccharide (LPS) in the incubation medium of the bacteria was evaluated via toxicity of supernatant of Y. pestis for intact animals (infection-toxic shock) and mice sensitized by D-GalN. Results. 37°C cultures of Y. pestis EV76 containing a full amount of plasmids were established to release LPS into the environment. Non-plasmid variants of both vaccine and virulent strains of Y. pestis pMTl', pCD Г, рРСР 1 do not have this ability. Separation of LPS from cell wall was detected in live bacteria of plague infectious agent. This process is assumed to be coupled with translocation of proteins coded by pMTl, pCDl, pPCPl plasmids from the cell into the environment. Conclusion. Functional interconnection between extrachromosomal elements of heredity and toxic activity of Y. pestis LPS is established for the first time

<I>In vivo</I> Tolerance to Lipopolyssacharides of Vaccine and Virulent <I>Yersinia pestis</I> Strains

The endotoxin tolerance phenomenon has been reproduced in vivo. Different forms of lipopolysaccharide (LPS) of vaccine (EV 76) and virulent (231) Y. pestis strains, grown at 28 and 37 °C, and LPS of S- and R-chemotypes of Escherichia coli were injected to the white mice in different combinations. Determined is the fact that primary and repeated injection of plague microbe LPS preparations in any combinations results in suppression of inflammatory response regardless of the differences in the LPS structure. In case of combined administration of Y. pestis LPS and E. coli LPS, the response depends on the LPS form and can vary from complete or partial tolerance to its total absence