RESUMEN

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 vac- cine strain Y pestis EV76 (pMT1, pCD1, pPCP1) and non-plasmid variants of vaccine EV76 (pMT1⁻, pCD1⁻, pPCP1⁻) and virulent 231 (pMT1⁻, pCD1⁻, pPCP1⁻) 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 intactanimals (infec- tion-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 pMT-, pCD1-, pPCP1- 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 pMT1, pCD 1, pPCP 1 plasmids from the cell into the environment. CONCLUSION: Functional inter-connection between extrachromosomal elements of heredity and toxic activity of Y pestis LPS is established for the first time.

Asunto(s)

Proteínas Bacterianas/genética , Pared Celular/metabolismo , Lipopolisacáridos/genética , Plásmidos/genética , Factores de Virulencia/genética , Yersinia pestis/genética , Yersinia pestis/clasificación

RESUMEN

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 intracellular cycle of plague development, and pro-inflammatory cytokine hyper-production is observed at the terminal stage of infection-toxic shock.

Asunto(s)

Citocinas/inmunología , Inmunomodulación , Lipopolisacáridos/inmunología , Células Th2/inmunología , Receptor Toll-Like 4/inmunología , Yersinia pestis/inmunología , Animales , Humanos , Transducción de Señal/inmunología

RESUMEN

AIM: Comparative study of the effect of endotoxin tolerance of mice to the effect of lipopolysaccharide (LPS37) and complex of lipopolysaccharide with mice toxin (LPS37-MT) of a virulent Yersinia pestis 231 strain. MATERIALS AND METHODS: Preparations of LPS of highly virulent strain Y. pestis 231 obtained by phenol method from cells cultivated at 37 degrees C as well as commercial preparations of S-LPS and R-LPS of Escherichia coli were used. Mice toxin was isolated from vaccine strain Y. pestis EV76. Effect of endotoxin tolerance was determined in mice treated with aminosugar D-galactosamine. RESULTS: The effect of initial LPS37 and modified form LPS37-MT of Y. pestis 231 was established to significantly differ from each other. When Y. pestis LPS37 is combined with heterologous forms--E. coli LPS or Y. pestis LPS37-MT, the inflammatory response of the organism differs and varies from complete or partial tolerance to complete lack thereof. For LPS37-MT complex only the sequence of administration to bioassay animals of LPS preparations is principal. In the case when primary activation is carried out by LPS37-MT and secondary--by Y. pestis LPS37 or S- and R- forms of E. coli LPS--the tolerance effect is absent. On the contrary, if LPS37-MT is used for recurrent activation against the background of all the other LPS forms including Y. pestis 231 LPS37 the inflammatory response is completely suppressed. CONCLUSION: Tolerance of mice to effect of LPS and LPS-MT complex of virulent Y. pestis 231 strain was shown to be different.

Asunto(s)

Tolerancia Inmunológica , Lipopolisacáridos/inmunología , Yersinia pestis/inmunología , Animales , Lipopolisacáridos/toxicidad , Ratones

RESUMEN

AIM: To study toxicity of lipopolysaccharides (LPS28 and LPS 37) of Yersinia pestis for mice sensitized by D-galactosamine (D-GalN). MATERIALS AND METHODS: LPS were obtained by the Westphal method from Y. pestis EV76 strain grown at temperatures of 28 and 37 degrees C. Dexamethasone and pentoxifylline were used as immunodepressants. Uridine was used for interruption of D-GalN effect. RESULTS: It was revealed that administration of D-GalN to mice increased their sensitivity to LPS of Y. pestis. Maximal increase in LPS toxicity was observed after simultaneous administration of D-GalN and LPS. D-GalN in dose 20 mg per mouse determined 100% lethality of animals during 24 h after administration of 10 mcg of LPS28 and 25 mcg of LPS37. Uridine in dose of 20 mcg per mouse administered 1 h after LPS and D-GalN neutralized effect of LPS in the presence of D-GalN. Dexamethasone and pentoxifylline did not protect animals sensitized by D-GalN against lethal effect of Y. pestis LPS. CONCLUSION: It was found experimentally that D-GalN enhances toxic effect of LPS28 in hundreds of times, and non-toxic LPS37 of Y. pestis EV76 demonstrated toxicity comparable to LPS28. Thus the D-GalN model could be used for enhancement of laboratory animals sensitivity to effect of Y. pestis LPS.

Asunto(s)

Galactosamina/toxicidad , Lipopolisacáridos/química , Lipopolisacáridos/toxicidad , Yersinia pestis/química , Animales , Ratones

RESUMEN

AIM: Determine correlation between toxicity and cytokine inducing activity of parent and conformation modified forms of lipopolysaccharides (LPS) of virulent Yersinia pestis strain. MATERIALS AND METHODS: LPS was isolated by phenol method from Y. pestis 231 cells grown at 37 degrees C (LPS37). LPS37 was modified by "mice" toxin (MT) Y. pestis. Toxicity was controlled in mice. TNFalpha and IFNgamma cytokine production was determined by enzyme immunoassay. The study was performed in human monocytes U-937 cell line. TLR4 re-stimulation was performed after activation of monocytes by S-LPS and R-LPS of Escherichia coli. RESULTS: LPS37 conformation change of virulent Y. pestis 231 strain during formation of complex with "mice" toxin increases its toxicity for animals by 2 times. LPS37 and LPS37-MT induce TNFalpha and IFNgamma synthesis by human monocytes. LPS37 simultaneously activates MyD88-dependent as well as MyD88-independent signal pathways. Modified LPS37-MT form is a strong activator only of MyD88-dependent pathway and thereafter induces synthesis of predominately one of the cytokines--TNFalpha. Monocyte response to primary and recurrent activation by LPS37 and LPS37-MT corresponds to R- and S-LPS E. coli cytokine response profile. CONCLUSION: A direct correlation between toxicity of LPS37 and LPS37-MT and their TNFalpha-inducing activity was demonstrated in the study. LPS37 and LPS37-MT of Y. pestis 231 differentially activates TLR4 signal pathways of human monocytes.

Asunto(s)

Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Monocitos/inmunología , Transducción de Señal/inmunología , Yersinia pestis/inmunología , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/farmacología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/química , Glicoconjugados/química , Glicoconjugados/inmunología , Glicoconjugados/farmacología , Humanos , Interferón gamma/biosíntesis , Dosificación Letal Mediana , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Ratones , Ratones Endogámicos , Monocitos/efectos de los fármacos , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Yersinia pestis/química , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidad

RESUMEN

UNLABELLED: AIM. To study dynamics of synthesis of TNF-alpha and INF-gamma by cell line U-937 human monocytes under the effect of Yersinia pestis EV 76 lypopolysaccharides (LPS) with different levels of toxicity: original LPS28 and LPS37 as well as their conformationally--changed variants with enhanced toxicity--complex of LPS with murine toxin (MT) of Y. pestis, and LPS modified by biologicall active compound (BAC) obtained from human erythrocytes. MATERIALS AND METHODS: Using phenol method, LPS were obtained from Y. pestis EV 76 cells grown at 28 and 37 degrees C. Production of cytokines was measured by ELISA. RESULTS: It was shown that original and modified forms of LPS28 and LPS37 induce synthesis of both TNF-alpha and INF-gamma by human monocytes. Expression of genes for two ways of synthesis of these cytokines points to activation and transmission of signal induced by all studied forms of Y. pestis EV 76 LPS through TLR4. Levels of activity of MyD88-dependent and MyD88-independent signaling pathways are different and depend from chemical structure of LPS28 and LPS37, conformation of their modified forms and duration of their exposition with monocytes. Dynamics ofcytokine synthesis corresponds to response of synergized TLR on activation with profound agonistic/antagonistic effect. CONCLUSION: It was determined that conformational modifications of Y. pestis EV76 LPS occurring due to effect of MT and BAC accompanied by quantitative, qualitative and temporal changes of TNF-alpha and INF-gamma synthesis by human monocytes and correlate with increase of their toxic properties.

Asunto(s)

Interferón gamma/biosíntesis , Lipopolisacáridos/inmunología , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Yersinia pestis/inmunología , Animales , Factores Biológicos/farmacología , Células Cultivadas , Eritrocitos/química , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/toxicidad , Ratones , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Temperatura , Receptor Toll-Like 4/metabolismo , Yersinia pestis/crecimiento & desarrollo

RESUMEN

Y. pestis "mouse" toxin and endotoxin have been found to be capable of being activated with hemolyzed mammalian red blood cells. The LD50 of the activated endotoxin decreases 5-10 times in comparison with the initial preparation. The LD50 of the activated "mouse" toxin decreases 5 times. As revealed in this study, the joint introduction of nonlethal doses of "mouse" toxin and endotoxin is highly toxic for white mice and guinea pigs. The presence of both "mouse" toxin and endotoxin in the toxic mixture is an essential factor for these two species of animals.

Asunto(s)

Toxinas Bacterianas/toxicidad , Endotoxinas/toxicidad , Eritrocitos/microbiología , Hemólisis , Yersinia pestis , Animales , Toxinas Bacterianas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Endotoxinas/aislamiento & purificación , Escherichia coli , Glucolípidos/aislamiento & purificación , Glucolípidos/toxicidad , Cobayas , Humanos , Dosificación Letal Mediana , Ratones , Plásmidos , Proteínas Recombinantes/toxicidad

RESUMEN

A method was elaborated for elimination of plasmids from the plague microbe by aminazine. A set of isogenic derivatives of the vaccine strain Yersinia pestis EV76 with different plasmid profiles has been obtained.

Asunto(s)

Clorpromazina/farmacología , Plásmidos/efectos de los fármacos , Yersinia pestis/genética , Genes Bacterianos

RESUMEN

The study has revealed that for the utilization of iron contained in transferrin the direct contact of Y. pestis with this metalloprotein is necessary. At 28 degrees C Y. pestis utilizes iron contained in transferrin. At 37 degrees C Y. pestis absorbs transferrin, but cannot utilize its iron, which is probably linked with disturbances in the system of the transfer of iron from the transferrin receptor complex into the bacterial cell.

Asunto(s)

Hierro/metabolismo , Temperatura , Transferrina/metabolismo , Yersinia pestis/metabolismo , Absorción , Transporte Biológico , Medios de Cultivo/metabolismo , Escherichia coli/metabolismo , Radioisótopos de Hierro , Unión Proteica

RESUMEN

Four Y. enterocolitica strains (10166, 10373, 2119, 5513) have been studied for the presence of the enzymatic systems of modification-restriction (M-R). As revealed with the use of cross titration, strains 10166 and 10373 contain M-R systems, supposedly of type II.

Asunto(s)

Enzimas de Restricción del ADN/clasificación , Yersinia enterocolitica/enzimología , Adsorción , Lisogenia , Especificidad de la Especie , Fagos T , Yersinia enterocolitica/genética

RESUMEN

Using enzymatic modelling of in vitro methylation of chromosome DNAs from Yersinia pestis EV 76, E. coli 834 and E. coli C600 RII by DNA methylases of Eco RII and Eco dam as well as of DNA hydrolysis of plasmid pBR 322 from the cells of Y. pestis EV 76, E. coli C600 and E. coli 834 by restrictases of Eco RII and Cfu I, it was found that cytosine DNA methylase from plague bacteria does not correspond to the type of RII methylases of E. coli. Adenyl DNA methylase is related to E. coli methylases type dam and modifies adenine in the nucleotide sequence of GATC.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Metiltransferasas/metabolismo , Yersinia pestis/enzimología , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Hidrólisis , Técnicas In Vitro , Metilación , Metiltransferasas/genética , Plásmidos , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica) , Yersinia pestis/genética

RESUMEN

An attempt at revealing the system of host specificity in Y. pestis by the methods of transformation and conjugation has been made. No endonuclease restriction has been detected.

Asunto(s)

Yersinia pestis/patogenicidad , Conjugación Genética , ADN Bacteriano/genética , Farmacorresistencia Microbiana , Escherichia coli/genética , Plásmidos , Transformación Bacteriana , Yersinia pestis/genética

RESUMEN

DNA-methyltransferases of Yersinia pestis EV, plague agent bacteria were isolated by P-II phosphocellulose chromatography. The methylating activity is eluated by two fractions at the 0.47 M and 0.53 M NaCl concentrations. Methylases of the plague microbe are specific with respect to two bases (adenine and cytosine) and are capable of modifying both native and denaturated form of DNA.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metiltransferasas/metabolismo , Yersinia pestis/enzimología , Cromatografía de Afinidad , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Cinética , Desnaturalización de Ácido Nucleico , Especificidad por Sustrato