Characteristics of the action of the biologically active factor from virulent Shigella flexneri strains in a study on mice and a cell culture]

The authors subjected to further study the biologically active factor revealed by them earlier in the virulent Sh. flexneri cultures by using the genetically bound triad of Sh. flexneri 5a-222 cultures and the corresponding couple of Sh. flexneri 2a-516. There was shown correlation of the strains virulence determined by the keratoconjunctival test, with the presence of genetically-determined production of the biologically active factor detectable in the culture filtrate, which produced toxic action of the continuous cell cultures in the virulen Sh. flexneri strains of different serovars (2a and 5a), and lethal action in intravenous injection to mice. Comparative study of toxicity of the preparations of the endotoxin, free endotoxin, and neurotoxin types showed the biologically active factor to resemble the neurotoxin, differing from it in the toxic action and thermolability. Filtrates of the virulent and genetically characterised avirulent strains differed in the protein and lipids content, this permitting to suggest participation of the protein and lipid complex in the toxic action of the biologically active factor.     

Biological activity of extracts isolated from a virulent strain of Sh. flexneri grown in the presence of calcium ions]

The authors carried out a comparative study of the genetically connected Sh. flexneri cultures (3 virulent strains, 3 clones of an avirulent mutant selected in the flux of an oblique light from the virulent strain, and lac+ Kcp A-hybrids obtained by crossing the initial virulent cultures with the E. coli K12 Hfr strains). The absence of any correlation between the virulence of the strains under study and the lipopolysaccharide (by rhamnose) content in the extracts from them in growing the cultures in the presence of calcium ions was noted. Toxicity of the extracts from the virulent cultures was demonstrated on a model of developing chick embryos. No such property was possessed by the extracts from avirulent strains. The extracts from the virulent cultures in nontoxic doses possessed the capacity to decrease LD50 of shigella strains used for the infection. The biologically active factor determined in the extracts from the virulent cultures apparently was not lipopolysaccharide.     

Analysis of merodiploid strains of Sh. flexneri that have lost virulence]

Episome F'13 introduced into the genome of a virulent Sh. flexneri strain brought about changes in a number of properties of the recipient strain. The expression of these properties was not connected with the chromosome area allelic to the plasmid genome. These changes seem to be induced by the mobilization of the chromosome genes of E. coli. The loss of virulence in Sh. flexneri strains carrying episome F'13 seemed to be the consequence of two reasons: the overlapping of kcpA gene by its dominant avirulent allele and abnormal synthesis of cell wall lipopolysaccharide due to the transfer of the mobilized genes from the donor strain F'13. When the preliminary mapping of genes on the chromomome was made with the use of plasmids, it was found necessary to use F-episomes which had no influence on the changes occurring in the phenotypic characteristics of the recipient.     

The role of delayed hypersensitivity in the development of an experimental dysentery infection and vaccinal process

The results of experiments, indicating the development of cell-mediated delayed hypersensitivity (DH) skin reaction, its dependence on the virulence of Shigella flexneri 2a, the dose and the character of the antigen used for testing the reaction, are presented. One of the immunologically active components of S. flexneri virulent strain 2a No. 516 is a biologically active thermolabile factor detected in filtrate and supernatant fluid. These data suggest that the immunosuppressive action of the virulent strain is probably linked with the function of T-suppressors affecting the reaction at the period of its development (the induction phase). S. flexneri avirulent strain produced no such effect, developing DH being seemingly sufficient for activating T-cell-mediated immune response.     

Increased sensitivity of immune macrophages to the cytotoxic action of virulent shigellae

The in vitro interaction of live bacteria belonging to virulent and avirulent Shigella and Salmonella strains with peritoneal macrophages obtained from mice immunized by the intragastric administration of these bacteria has been studied. In contrast to Salmonella-activated macrophages capable of resisting the intracellular proliferation and the cytopathic action of homologous bacteria, Shigella-activated macrophages become more sensitive to the cytopathic action of virulent shigellae. The ability of shigellae to render an aggravating cytopathic effect on the activated macrophages correlates with the virulence of dysentery bacilli and is practically absent in avirulent strains, including S. flexneri 2a No. 516 M vaccine strain.     

Interrelation of virulent and avirulent (vaccinal) strains of Shigella flexneri 2A in long-term bacterial carriage in gnotobiotic rats

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.     

Electron microscopic study of the interaction of virulent and avirulent Shigella flexneri strains with the macrophages of murine peritoneal exudate

The electron microscopic dynamic study of the interaction of S. flexneri virulent strain 2a and S. flexneri avirulent strain, genetically related to strain 2a and having the same antigenic structure, with peritoneal macrophages of CBA mice at an early period showed differences in the cytotoxic action of these strains. Even at the early period of the interaction between macrophages and virulent shigellae structural changes were observed in macrophages: ruptures in the membranes of parasitophore phagosomes, the vacuolization of the cytoplasm, the destruction of mitochondria. These changes were indicative of the early cytotoxic action of shigellae belonging to the virulent strain. In macrophages infected with avirulent shigellae the manifestations of this cytotoxicity were less pronounced and appeared later than in macrophages infected with S. flexneri virulent strain.     

Experimental study, using a pulmonary model, of the pathogenic properties of genetically related strains of Sh. flexneri, differing in their ability to cause keratoconjunctivitis]

Complex microbiological and morphological study of the genetically connected Sh. flexneri strains, avirulent by keratoconjunctival test showed that the greatest extent of the virulent loss was revealed in shigellae with mutation in the area glpK-gene: they lost the capacity to penetrate into the cells of the bronchial epithelium, to resist local leukocytic reaction and to produce an injurious action on the pulmonary vessels. Shigellae with the replaced kcpA- gene area mostly lost the capacity to penetrate into the epithelial cells of mouse bronchi and were eliminated from the epithelium by the 9th hour after the administration. Xyl--avir-hybrids obtained from crossing of Sh flexneri with the streptomycin-resistant E. coli K12 donors were characterized by the loss of the capacity to prolonged reproduction in the organism of mice with the retention of the capacity to penetration into the epithelium, of the influence on the development of the leukocytic reaction and of the damaging effect on the pulmonary capillaries.     

Functional macrophage activity in infection with virulent and avirulent strains of the dysentery microbe

The effect of S. flexneri virulent and avirulent (vaccine) strains 2a on the cytoplasmic membrane of mouse macrophages has been studied by evaluating the action of these bacteria on the activity of 5-nucleotidase. The dynamics of the activity of 5-nucleotidase after the introduction of both virulent and avirulent strains has a phasic character with alternating rises and falls in the activity of this enzyme in comparison with the control. S. flexneri vaccine strain produces mainly a stimulating effect on the functional activity of peritoneal macrophages in mice, which is confirmed by a decrease in the activity of 5-nucleotidase; on the contrary, S. flexneri virulent strain- has mainly an inhibiting effect on the functional activity of peritoneal macrophages, which is confirmed by an increase in the activity of 5-nucleotidase in these cells. The comparative study of changes in the activity of 5-nucleotidase, following the introduction of S. flexneri, in mice, previously immunized with smallpox vaccine, and in intact mice has shown that the use of animals immunized with smallpox vaccine in the study of metabolic characteristics may lead to distortions in the results of the experiment.     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

Reaction of the hematopoietic system in mice to the administration of virulent and avirulent strains of Shigella flexneri 2A

The influence of live bacteria and antigenic preparations of virulent and avirulent stains of Shigella flexneri 2a on the endogenous splenic colony formation in murine hematopoietic cells has been studied. The different stimulating activity of live microbial cells of virulent and avirulent strains Shigella flexneri 2a and their antigenic preparations on the endogenous colony formation has been shown. The effect observed depends on the preparation doses and time of their administration before irradiation of the animals. The stimulating influence on the development of hemopoiesis endogenous foci may be conditioned by the action of heat-labile products of the live microbial cells and closely correlates with the virulence of strains studied.     

Surface components of shigellae involved in adhesion and haemagglutination

Strains of Shigella species were studied for their ability to adhere and agglutinate mammalian erythrocytes. Shigella dysenteriae and Sh. flexneri exhibited haemagglutinating (HA) properties when cultured in Casamino Acids-Yeast Extract (CYE) broth in the presence of 1 mmol 1^-1 calcium chloride, but other shigellae did not show this property under the same culture conditions. Repeated subcultivation of Sh. boydii, Sh. sonnei and HA negative strains of Sh. dysenteriae and Sh. flexneri in CYE broth medium induced adhesive and haemagglutinating properties that were inhibited by sodium periodate. HA activities of Shigella spp. were also inhibited by N -acetylneuraminic acid, α₁-glycoprotein and fetuin, but not by protease. Electron microscopy of Sh. dysenteriae 1, Sh. flexneri 2a, Sh. boydii 12 and Sh. sonnei 1 grown in CYE broth showed the presence of an extracellular slime layer that promoted agglutination of erythrocytes. The slime layer extracted from the cell surface of Shigella spp. showed HA properties, whereas lipopolysaccharide (LPS) obtained from the same strains, except Sh. dysenteriae 1, did not agglutinate erythrocytes. This evidence suggests that the cell surface haemagglutinin is a loosely bound slime layer which is expressed in CYE broth medium.     

Increase of capsular material thickness following in vivo growth of virulent Streptococcus suis serotype 2 strains

Abstract Protein profile and capsular material thickness of Streptococcus suis serotype 2 strains were compared after in vitro and in vivo growth. Three virulent and one avirulent strains were used. These strains were grown in Brain Heart Infusion (BHI) broth, cells were collected by centrifugation, resuspended in a sterile saline solution and injected in diffusion chambers. The devices were then inserted in rat abdomens for 17 h. In vitro grown strains were also inoculated into fresh BHI broth and cultivated for 17 h at 37°C. In vivo as well as in vitro grown bacteria were harvested by centrifugation, processed in a French pressure cell, treated with lysozyme and centrifuged to collect cell proteins for SDS-PAGE analysis. Transmission electron microscopy using polycationic ferritin labeling to stabilize capsular material was also carried out. No significant modification was noted in the protein profile for any strain after in vivo growth except for a 39 kDa protein of one virulent strain. On the other hand, an increase in thickness of capsular material was noted for the three in vivo grown virulent strains while no change was noted for the avirulent strain. This increase in capsular material thickness of virulent strains was accompanied by an increased resistance to killing by pig polymorphonuclear leukocytes. The capacity to produce more capsular material in vivo seems to be an attribute of some virulent S. suis serotype 2 strains.     

Differentiation of virulent strains of Shigella and entero-invasive Escherichia from their avirulent variants using modified indirect immunoenzyme analysis

The data obtained in this investigation confirm that the modified indirect enzyme immunoassay (EIA) permits the differentiation of virulent bacteria of the genus Shigella and enteroinvasive Escherichia (group 1), regularly containing virulence plasmids with a molecular weight of 120-140 MD, from their avirulent variants which have lost these plasmids (group 2). The ratio of the optic density (OD) values of the positive control samples (the OD of group 1) to the OD values of the negative ones (the OD of group 2) is significantly higher than 2.1. As revealed by EIA, the differences between groups 1 and 3 (avirulent Shigella strains and E. coli smooth strain O124, retaining high-molecular plasmids with a molecular weight of 120-140 MD or their fragments) are statistically insignificant. The ratio of the OD of group 1 to the OD of group 3 is significantly less than 2.0. Analysis of outer membrane protein (OMP) preparations isolated from S. flexneri virulent strain 2a and its isogenic avirulent plasmid-containing variant has revealed significant differences in their EIA results. The ratio of the OP of OMP preparations isolated from the virulent strain to the OD of OMP preparations from the avirulent strain exceeds 2.1.     

Selection of bacterial wilt-resistant tomato through tissue culture

Bacterial wilt-resistant plants were obtained using a tomato tissue culture system. A virulent strain ofPseudomonas solanacearum secreted some toxic substances into the culture medium. Leaf explant-derived callus tissues which were resistant to these toxic substances in the culture filtrate were selectedin vitro and regenerated into plants. These plants expressed bacterial wilt resistance at the early infection stage to suppress or delay the growth of the inoculated bacteria. On the other hand, complete resistance was obtained in self-pollinated progeny of regenerants derived from non-selected callus tissues. These plants showed a high resistance when inoculated with this strain, and were also resistant when planted in a field infested with a different strain of the pathogen.     

Models for the study of the toxic action of Legionella pneumophila

L. pneumophila virulent culture and the filtrate of this culture disintegrated with ultrasound were shown to be toxic for guinea-pig peritoneal macrophages in vitro and for AKR mice. The virulent culture of this infective agent and the filtrate of the supernatant fluid obtained from the washings of its virulent culture had no toxic effect on a macrophage monolayer culture and on mice. The use of these models for characterizing Legionella intracellular toxic activity, as well as for characterizing Legionella strains isolated from patients and the environment, is proposed.     

Study of the role of type-specific antigen in the process of Shigella flexneri interaction with epithelial cell and macrophage cultures]

Epithelial cells of the HEp-2 line infected in parallel by genetically connected strains of Sh. flexneri, differing in the capacity to synthesize the type antigen were studied morphologically. The type antigen proved to show no significant influence on the penetration of dysentery bacilli into the cell. A study of the process of phagocytosis of the mentioned strains by a culture of peritoneal macrophages of guinea pig demonstrated that the presence of a type antigen communicated to the bacteria some selective advantages within the macrophages increasing their resistance to the action of the digestive enzymes.     

Intraspecies antagonism of Sh. flexneri in an HEp-2 cell line model]

The authors describe an effect of suppression of invasion of the guinea pig eye conjunctiva and the HEp-2 epithelial cells by virulent Sh. flexneri bacilli, with a simultaneous administration of the same dose of avirulent shigella mutants, genetically connected with them. The data of morphological study and experiments with 3H-glucose labeled shigellae carried out on the cell species model indicated that the bacterial competition for the specific sites for absorption on the epithelial cells underlay the observed phenomenon.     

弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

Relationship between adenylate cytokinin production and Ti plasmid of Agrobacterium tumefaciens

To know whether the tumor-inducing plasmid of Agrobacterium tumefaciens carries genetic information of the biosynthesis of cytokinins, the levels of 6-(3-methyl-2-butenyl-amino)purine (iPAde) and its 4-hydroxy derivative trans-zeatin (trans-Z) and its p-beta-D-ribofuranoside (trans-ZR) produced in media by wild-type virulent strain, plasmid-cured avirulent strain and the deletion mutant were compared. The highest levels of iPAde and trans-Z were found in the culture filtrate of late-log phase growth of plasmid-containing virulent strain, then the levels of iPAde and trans-Z were reduced rapidly at stationary phase. The plasmid-cured avirulent strain and deletion mutant had low levels of iPAde and trans-Z throughout the growth. Results obtained here showed Ti plasmid plays an important role in cytokinin biosynthesis.