The heterogeneity of the alveolar macrophages of guinea pigs immunized subcutaneously with a vaccinal strain of Yersinia pestis]

The study of alveolar macrophages obtained from guinea pigs at different periods after the subcutaneous immunization with Y. pestis vaccine strain has revealed the heterogeneity of these macrophages in their bactericidal activity with respect to Y. pestis and in their resistance to the cytopathic action of this microorganism, as well as in the content of lysosomes and in the expression of receptors on the cell surface. The study has shown that subcutaneous immunization does not lead to the redistribution of subpopulations and to changes in their digestive activity.     

The phagocytic activity of peritoneal macrophages in relation to Yersinia pestis with defective and complete Fra genes

In vitro study of phagocytosis has shown that in guinea pigs fraction 1 is conducive to the ingestion of Y. pestis by macrophages, to survival and proliferation of Y. pestis cells in these macrophages, as well as to their specific transformation leading to their increased ingestive and bactericidal activity with respect to Y. pestis. In mice the role of fraction 1 in phagocytosis has proved to be less significant.     

The properties of the cellular surface of Yersinia pestis strain EV and its achromogenic variants

The comparative study of the properties of the surface of vaccine strain Y. pestis EV and its achromogenic variants (AV) differing from the initial strain by decreased immunogenicity and by the morphology of colonies, has been made. The achromogenicity of Y. pestis colonies has been shown to correlate with the loss of the outer membrane protein with a molecular weight 22 kD. The synthesis of this protein is determined by chromosomal genes. AV have been found to have different sensitivity to bacteriophages. The analysis of the electrokinetic potential of Y. pestis EV and its AV has revealed that in the latter have surface charge is considerably greater (1.4- to 1.5-fold). As shown in this study, the hemagglutinating activity of AV with respect to red blood cells of humans with blood group I (O) and guinea pigs is decreased by 1-2 orders and these strains do not agglutinate with sheep red blood cells. The low activity of the initial stage of the phagocytosis of AV by mouse macrophages has been shown. The possible role of the 22 kD proteins as an adhesion factor is discussed.     

Changes in the antimicrobial potential of the phagocytosing cells of great gerbils in experimental plague

The work deals with the results obtained in the study of the activity of the cytocidal systems of phagocytes in great gerbils under normal conditions and during the plague infectious process. The data have been analyzed in comparison with the corresponding characteristics of phagocytic activity in mice. Changes in the oxygen-dependent metabolism (ODM) of neutrophils and macrophages, responsible for the functioning of their oxygen-dependent cytocidal systems, in plague show features characteristic of the nonspecific systemic postaggression reaction (SPAR). In Y. pestis-sensitive animals (mice), changes in the ODM activity of phagocytes are manifested as shock of different severity. In great gerbils, these changes present as true SPAR. The degree of the sensitivity of animals to Y. pestis infection is mainly determined by the initial level of the activity of oxygen-sensitive cytocidal systems of neutrophils (determined in the nitro blue tetrazolium spontaneous reduction test). In great gerbils this level exceeds the corresponding characteristics in mice 2.6- to 8.7-fold.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

Interleukin-1-inducing activity of the polysaccharide-containing antigens of the cell wall in Yersinia pestis

The induction of the synthesis of interleukin-1 (IL-1) in human monocytes under the influence of the endotoxic preparations (LPS) and Y. pestis basis somatic antigen has been experimentally studied. The results obtained in this study make it possible to come to the conclusion that the capacity of the endotoxin of Y. pestis cell wall, consisting of LPS of type R, for inducing the synthesis of IL-1 in human monocytes is not different from the corresponding capacity of Salmonella and Shigella LPS, type S. Y. pestis O-specific polysaccharide in a discrete state has considerably greater IL-1-inducing activity in comparison with other preparations used in this experimental study. Such typical changes, characteristic of the initial stage of Y. pestis infection, as a sharp rise in temperature, transitory neutropenia, significant primary suppression of the oxygen-dependent metabolism of polymorphonuclear neutrophils are probably due to the induction of the synthesis of IL-1 by the polysaccharide-containing antigen of Y. pestis cell wall (LPS, basic somatic antigen) in cells of the mononuclear phagocytizing system.     

Comparative evaluation of mono- and neutrophilokins inducing activity of Yersinia pestis antigens

The results of the comparative analysis of the cytokine inducing activity of Yersinia pestis EV antigens are presented. Y. pestis fraction 1A (F1A) and lipopolysaccharide (LPS) were shown to induce mono- and neutrophilokines, regulating cooperative interaction of phagocytes in the process of immunity formation to plague. Neutrophilokines and monokines exceed in their capacity for inducing F1A such acknowledged inductor as Escherichia coli LPS. As revealed by the comparative evaluation of Y. pestis EV LPS and E. coli LPS, neutrophilokines synthesized under the action of the former preparation, have greater influence on the inhibition of the macrophage migration from the infection focus as well as on digestive activity of these cells (in secondary immune response) and on the labilization of the lysosome membranes of macrophages than neutrophilokines induced by E. coli LPS. At the same time they produce a lesser modulating effect on the killer and chemotactic activity of neutrophils, as well as on the expression of FC receptors (FcR) on their surface in comparison with monokines, synthesized under the influence of E. coli LPS.     

The effect of the adhesion pili of Yersinia pestis on the physiological activity of the leukocytes and macrophages in experimental animals

The data on the influence of the preparation of Y. pestis adhesion pili on peritoneal macrophages in white mice and guinea pigs are presented. Y. pestis adhesion pili have been found to induce the dose-dependent increase of cell chemiluminescence. They have also been found to induce a number of biochemical changes in target cells: the secretion of myeloperoxidase, an increase in the activity of cAMP-dependent protein kinases.     

Plasminogen activator Pla of Yersinia pestis utilizes murine DEC-205 (CD205) as a receptor to promote dissemination

Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (MPhis) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infection. Both alveolar MPhis and pulmonary dendritic cells express a C-type lectin receptor, DEC-205 (CD205), which mediates antigen uptake and presentation. However, no ligand has been identified for DEC-205. In this study, we show that the invasion of alveolar MPhisby Y. pestis depends both in vitro and in vivo on the expression of PLA. DEC-205-expressing MPhis and transfectants, but not their negative counterparts, phagocytosed PLA-expressing Y. pestis and Escherichia coli K12 more efficiently than PLA-negative controls. The interactions between PLA-expressing bacteria and DEC-205-expressing transfectants or alveolar MPhis could be inhibited by an anti-DEC-205 antibody. Importantly, the blockage of the PLA-DEC-205 interaction reduced the dissemination of Y. pestis in mice. In conclusion, murine DEC-205 is a receptor for PLA of Y. pestis, and this host-pathogen interaction appears to play a key role in promoting bacterial dissemination.     

Changes in the oxygen-dependent metabolic activity of polymorphonuclear leukocytes from the peripheral blood and macrophages from the peritoneal exudate in the experimental infection of mice with the causative agent of plague

The subcutaneous infection of C57BL/6J mice and noninbred white mice with 40 LD100 of Y. pestis virulent strain has been found to produce synchronous changes in the oxygen-dependent metabolism (ODM) of peripheral blood neutrophils in the spontaneous or zymosan-, E. coli- and Y. pestis-stimulated variants of the NBT test. These changes can be divided into three phases: (I) the phase of a sharp drop in ODM activity; (II) the phase of the increase of this activity, occurring simultaneously with the penetration of Y. pestis cells into the blood stream; and (III) the phase of the terminal decrease of ODM activity as the cytotoxic lesion of phagocytic cells occurs. Peritoneal exudate macrophages show a more gradual decrease in ODM activity. The infection of the animals with 40,000 LD100 of Y. pestis has been found to produce an increase in the ODM activity of neutrophils, rapidly followed by its decrease to the zero level. Macrophages show phasic changes in their ODM activity, identical to changes in the ODM values of neutrophils in mice infected with 40 LD100 of Y. pestis.     

Results of a trial of a plague monoclonal antibody erythrocyte diagnostic agent

Plague antibody monoclonal erythrocyte diagnosticum was studied in serological tests simultaneously with commercial plague antibody erythrocyte diagnosticum prepared on the basis of hyperimmune horse serum and with commercial plague antigenic erythrocyte diagnosticum. In this investigation the suspensions of numerous strains of Yersinia pestis, other closely related and heterologous organisms, experimentally infected wild and laboratory animals, as well as samples of materials obtained from small rodents caught in several natural foci of plague, were studied. The monoclonal diagnosticum was, practically, not inferior to the similar commercial preparation with respect to the frequency of positive results and the activity of the materials under study in serological tests, but showed greater specificity, as it reacted strictly with Y. pestis capsular antigen.     

The phenomenon of pilus formation in the interaction of Yersinia pestis with macrophages in experimental animals

According to the data of the enzyme immunoassay, adhesion pili were expressed by Y. pestis cells EB76 after their cultivation in media with pH similar to that of phagolysosomes. The expression of adhesion pili was found to occur 18 hours after the contact of apiliate cells EB76 with a monolayer of native, but not inactivated (at 56 degrees C for 30 minutes) macrophages of the peritoneal exudate of white mice and guinea pigs. Purified adhesion pili possessed cytotoxic action and inhibited the digestive activity of macrophages with respect to Y. pestis. The formation of pili in interaction with macrophages and the pronounced effect of the preparations of purified pili on the function of phagocytes make it possible to regard the formation of pili as an important determinant of virulence.     

Production of tetanolysin preparations and characteristics of their properties

As the result of the study of tetanolysin-producing Clostridium tetani strains, their populations have been found to be markedly heterogeneous with respect to the hemolytic activity of clone cultures. On the basis of normal and dialyzed cultures of selected variants with maximum activity the preparations of tetanolysin have been obtained, and their hemolytic activity and antigenic properties have been studied. Antihemolytic rabbit sera have also been obtained and characterized. Partially purified preparations of tetanolysin with high hemolytic activity have been obtained by the fractionation of C. tetani dialyzed cultures with ammonium sulfate.     

Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow-derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis-infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.     

Experimental evaluation of interaction between Yersinia pestis and soil infusoria and possibility of prolonged preservation of bacteria in the protozoan oocysts

The character and outcome of interactions between Y. pestis (vaccine strain and soil infusoria Tetrahymena pyriformis (axenic culture) were under experimental study. The parallel use of the bacteriological method and PCR test systems made it possible to follow the dynamics of Y. pestis cells (strain EV) with different plasmid profiles in their interaction with infusoria, as well as their passage into the protozoa cysts. The study revealed the complete utilization of Y. pestis cells lacking virulence factors by infusoria. The presence of plasmids of virulence influenced only the duration of complete bacterial phagocytosis. A drop in the temperature of cultivation to 2 degrees C induced the mass and rapid encystment of infusoria. In the PCR analysis specific DNA fragments of Y. pestis cells, preserved in the latent (uncultivable) state, were detected in the cysts of protozoa within the period of up to 14 months, while the results of bacteriological studies were negative. The data thus obtained are discussed with regard to the possible mechanisms of the existence and prolonged reservation of Y. pestis in the soils of natural foci with participation of protozoa.     

Inhibition of natural killer activity by human bronchoalveolar macrophages

Mononuclear phagocytes were isolated by adherence from peripheral blood, peritoneal exudates, early lactation milk, ovarian carcinomatous ascites and bronchoalveolar lavages. Their capacity to modulate natural killer (NK) activity was assessed by mixing them with blood lymphocytes and by measuring lysis of 51Cr-labeled K562 cells. Unlike other mononuclear phagocyte populations, alveolar macrophages caused a marked dose-dependent inhibition of NK activity. Significant inhibition (40%) of the expression of cytotoxicity was evident at a ratio of alveolar macrophages to lymphoid cells of 0.12:1, and more than 80% suppression was usually observed at a ratio of 0.5:1. Blood monocytes, peritoneal and milk macrophages were consistently inactive up to the highest ratio tested, 2:1. Inhibition of the expression of NK activity by alveolar macrophages was observed at lymphocyte to K562 ratios ranging from 6:1 to 100:1 and over a 4 h or 20 h 51Cr release assay. Alveolar macrophages also inhibited interferon-stimulated cytotoxicity. Alveolar macrophages are unique among the mononuclear phagocyte populations studied in their capacity to inhibit the expression of NK activity effectively, and they could play a role in determining the low levels of NK activity associated with human pulmonary tissue.     

The mutagenic effect of polymorphonuclear leukocytes on Yersinia pestis

As the result of in vitro experiments, Y. pestis auxotrophic mutants have been obtained under the influence of polymorphonuclear lymphocytes obtained from guinea pigs, previously immunized with Y. pestis strain EV. The mutagenic effect has been found to occur at minute 45 of phagocytosis. The control treatment of the bacteria with the lysate of neutrophils, homologous serum, penicillin (antibiotic-influenced selection) has not been found to lead to the appearance of auxotrophicity. These data suggest that the polymorphonuclear leukocytes of animals, having a set of powerful cytocidal systems, play an active role in the process of the natural variability of Y. pestis.     

Numerical analysis of the phenotypic properties and total genomic characteristics of strains of Yersinia pestis related to different subspecies

The numerical analysis of the phenotypical properties of Y. pestis strains, classified with 5 subspecies by 60 signs, was carried out. In comparing the properties of strains belonging to the main (nomenclature) subspecies with strains of other subspecies, the similarity index varied within the range 82-95%. A high degree of genetic affinity between 21 Y. pestis strains of five subspecies was demonstrated by the method of molecular DNA-DNA hybridization. The level of DNA homology with respect to the alpha-CTP.[3H] reference mark of Y. pestis P-1300 in strains belonging to different subspecies was found to be 84-97%. The plasmid spectrum of 25 examined strains of these three subspecies proved to be identical and consisted of plasmids similar in their electrophoretic motility to marker plasmids from Y. pestis strains EV from the Research Institute of Epidemiology and Hygiene and Otten.     

Thymine- and thymidine-dependence of Yersinia pestis mutants with altered thymidylate synthetase

Thymine- and thymidine-dependent mutants of Y. pestis strain EV-76 have been isolated and characterized. Obtaining Y. pestis thymine-dependent mutants in trimethoprim-containing media with full nutritional value in the presence of thymine and thymidine and the capacity of natural strains from the foci of infection in Transcaucasia and Mongolia to grow in such media indicate that Y. pestis has gene tpp controlling thymidine phosphorylase, but this enzyme is strongly suppressed under normal conditions. The capacity for its suppression under definite conditions and the degree of the activation of thymidine phosphorylase determine the realization of Thy and Thyd phenotypes in Y. pestis mutants under study, though both types of these mutants have a mutation damage of gene thy A coding the synthesis of thymidylate synthetase.     

Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.