YscU recognizes translocators as export substrates of the Yersinia injectisome.

YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

Type III secretion gets an LcrV tip.

Type III secretion is used by many Gram-negative pathogenic bacteria to inject effector proteins into eukaryotic host cells. Effector delivery requires a secretion apparatus, called an injectisome or needle complex, and the assembly of a translocation pore in a target-cell membrane. Recent work provides evidence that enlightens the view of how pore assembly might occur and of how the injectisome and the pore might be linked.

[Electron microscopy study of bacterial adhesiveness]. / Elektronno-mikroskopicheskoe issledovanie adgezivnosti bakterii.

The interaction of Staphylococcus aureus, Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. frederikseni, Y. kristenseni and erythrocytes was studied with the use scanning electron microscopy. Highly adhesive and moderately adhesive Staphylococcus and Yersinia strains displayed both individual coated bacterial cells and groups of cells interconnected by common intercellular matrix on the surface of erythrocytes. In nonadhesive Staphylococcus and Yersinia strains no coating was detected on the surface of bacterial cells. Some of Staphylococcus and Yersinia cells interacting with erythrocytes were at the stage of heteromorphism with different manifestations of L-transformation (cells with cell wall defects, spheroplasts and protoplasts). Heteromorphic cells did not adhere to the surface of erythrocytes.

[Electron microscopic study of pathogenic bacteria on environmental objects]. / Elektronno-mikroskopicheskoe issledovanie patogennykh bakterii na ob"ektakh vneshnei sredy.

The morphological picture of different bacteria (Salmonella typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia enterocolitica O3, Y.pseudotuberculosis 1, Y.frederiksenii, Y.intermedia, Y.kristensenii) on environmental objects was studied with the use of scanning electron microscopy (SEM). Bacteria adhered to the surface of pieces of fodder, egg shell, cabbage leaves and form microcolonies, whose morphology was similar to colonies, grown on nutrient media. The cells produced extracellular substances, seen in SEM as integuments. These integuments were gourd to protect the population from the action of unfavorable factors.

Studies on the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species.

A total of 1242 strains of Y. enterocolitica, 104 strains of Y. frederiksenii, 95 strains of Y. kristensenii and 85 strains of Y. intermedia were serotyped with antisera against 56 O antigens and 19 H antigens according to the extended antigenic scheme of Wauters, and with additional antisera against 4 somatic and 19 flagellar antigens not previously described. H antigens of Y. frederiksenii, Y. kristensenii, and Y. intermedia turned out to be rather homogeneous without distinct subfactors. In these species the scope of identified serovars was narrow. Flagellar antigens of Y. enterocolitica were mostly composed of several subfactors, leading to a total of 117 serovars identified in the species. A number of cross-reactions between Yersinia H antigens were observed which could be avoided by absorption without significant lowering of the titre. Flagellar antigens of Yersinia were monophasic, and species specific. The antigens remained stable after storage in agar stabs and repeated subcultures. The epidemiological value of serotyping is demonstrated by strains from three different sources. It is suggested serotyping of Yersinia strains should be performed in three steps: O typing of the prevailing enteropathogenic Y. enterocolitica serogroups in the medical routine laboratory; O and H typing of Y. enterocolitica by National Reference Centres applying a typing scheme reduced to this species; and O and H typing of Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia by specialized International Centres using an extended typing scheme. The need for international standards comparable to those established for Salmonella is emphasized.

[Phenomenon of pili formation in Yersinia enterocolitica]. / Fenomen pileobrazovaniia u Yersinia enterocolitica.

In Y. enterocolitica strain, serovar 0:10, the capacity for the formation of pili inducing the mannose-resistant hemagglutination (MRHA) of formolated sheep red blood cells was due to the presence of plasmid pYE10. MRHA-inducing pili differed serologically from Y. pestis and Y. tuberculosis adhesion pili. Plasmid pYE10 was immobilized for transfer to cells of Escherichia coli strain HB101 (rec A) by means of pRP 4. The expression of MRHA-inducing pili in the new host the rec A-independent character of the synthesis. Y. enterocolitica cells containing pYE10 agglutinated in tissue-culture media with 10% of serum added at 37 degrees C.

Production of bacteriocin-like substances by Yersinia frederiksenii, Y. kristensenii, and Y. intermedia strains.

The production of bacteriocin-like substances by strains of Yersinia frederiksenii, Y. kristensenii and Y. intermedia in broth culture was established. These substances showed a selective activity against Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia strains. Electron micrographs revealed the presence of phage tails in culture media. The production of these substances was detected in cultures grown at 25 degrees C but not in those grown at 37 degrees C, while these bacteriocin-like substances were active at 25 and 37 degrees C. Y. enterocolitica serogroups 0:3 and 0:9 were more susceptible to these bacterin-like substances than strains of Yersinia isolated from environmental sources.

A novel virulence-associated cell surface structure composed of 47-kd protein subunits in Yersinia enterocolitica.

A fresh human isolate of Yersinia enterocolitica serotype 03, and its derivative that had lost the virulence-associated 46-Md plasmid, were grown under defined conditions and compared for their outer membrane protein and cell surface structure. Under these conditions, the virulent strain grown at 37 degrees C expressed one major outer membrane protein (47 kd) not present in the plasmidless strain or in either strain grown at room temperature. A 200-kd protein also seen in the same preparations was shown to be an oligomer composed of the 47-kd protein subunits. Four different electron microscopic techniques showed tack-like projections covering the surface of those bacteria that expressed the 47-kd protein. These were specifically labeled with antibody to the 47-kd protein. This surface structure appeared to mediate aggregation (auto-agglutination) of the bacteria bringing their surfaces into unusually close apposition.

Temperature-inducible surface fibrillae associated with the virulence plasmid of Yersinia enterocolitica and Yersinia pseudotuberculosis.

When cultivated at 37 degrees C in static broth, human clinical isolates of Yersinia enterocolitica (serogroups O:3, O:8, and O:9) and Yersinia pseudotuberculosis (serogroup O:III) produced numerous nonflagellar surface appendages, which appeared as a lawn of fine fibrillae, each having a diameter of 1.5 to 2.0 nm and a length of 50 to 70 nm. Cultivation at 22 degrees C resulted in complete disappearance of the fibrillae. The phenotypic expression of these appendages was correlated with the presence of the 40- to 48-megadalton virulence plasmid and was strongly affected by the growth medium. Evidence is presented which suggests that these plasmid-mediated, temperature-inducible surface fibrillae are responsible for autoagglutination and are related to production of one prominent, Sarkosyl-insoluble polypeptide of ca. 180 kilodaltons in the bacterial outer membrane.

[Adhesion of Yersinia pseudotuberculosis serotype III]. / Adgeziia Yersinia pseudotuberculosis serotipa III.

The use of the erythrocyte agglutination test for characterizing the properties of Y. pseudotuberculosis led to the detection of a highly adhesive strain belonging to serotype III and capable of forming pili at 37 degrees C. The adhesion of the cells provided with pili was completely inhibited by the mixture of gangliosides, specific antibodies, and the preliminary treatment of erythrocytes with neuraminidases sharply enhanced the effectiveness of adhesion. The adhesion pili consisted of protein subunits with a molecular weight of 16 800 daltons and had the isoionic point at pH 4.1.

[Electron microscopic study of the interaction of Yersinia pseudotuberculosis with macrophages and HeLa cells]. / Elektronno-mikroskopicheskoe izuchenie vzaimodeistviia Yersinia pseudotuberculosis s makrofagami i kletkami HeLa.

The comparative electron-microscopic study of early stages of the interaction of Y. pseudotuberculosis virulent strain (No. 282) with "professional" (macrophages) and "nonprofessional" (HeLa cells) phagocytes has been carried out. The character of the intimate mechanism of this interaction has been found to be essentially different. The common feature for both systems is the adsorption of bacteria and their penetration into cells due to phagocytosis. But the subsequent fate of Y. pseudotuberculosis is different. In HeLa cells they are isolated from the cytoplasm by multilayer membrane structures, thus remaining morphologically intact. In macrophages the destruction of the microbe in phagolysosomes occurs.

Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis.

Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudotuberculosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca2+ dependent growth at 37 degrees C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.

Influence of media on temperature-dependent motility test for Yersinia enterocolitica.

The influence of six widely used media on the motility of 100 isolates of Yersinia enterocolitica grown at 25 and 37 degrees C was investigated. Seven isolates were motile at 25 and 37 degrees C in all media, and the presence of flagellated cells was demonstrated by a flagellum stain. Six isolates were nonmotile at both temperatures in all media, and no flagella were observed. Identification schemes for Y. enterocolitica should reflect these possibilities.

Localization of enterobacterial common antigen in Yersinia enterocolitica by the immunoferritin technique.

Rabbits were immunized with the enterobacterial common antigen (ECA)-immunogenic strain Escherichia coli F470. ECA-specific antiserum was obtained by absorbing the resulting antisera with the genetically closely related ECA-negative strain E. coli F1283. These two strains also served as positive and negative controls in the localization study of ECA in Yersinia enterocolitica strain 75, smooth and rough forms (Ye75S and Ye75R), by the indirect immunoferritin technique. Cells of Ye75S grown at 22 degrees C showed no labeling with ferritin after treatment with the ECA-specific antiserum and subsequent ferritin-conjugated goat anti-rabbit antibodies. If the cells were grown at 40 degrees C, however, most of the cells showed weak ferritin labeling. At this higher growth temperature, the lipopolysaccharide of this strain contains less O-specific chains (6-deoxy-L-altrose), as was shown in a previous study. The rough mutant Ye75R, which lacks O-specific chains completely, showed denser labeling with ferritin. These results indicate that ECA on the cell surface of Ye75S is covered by O-specific chains of the lipopolysaccharide if grown at 22 degrees C and is therefore not accessible to ECA antibodies. It becomes accessible, however, when O-chains are lacking (R mutants) or when they are reduced in size or amount (growth at 40 degrees C).

Adherence of fimbriate and non-fimbriate strains of Yersinia enterocolitica to human epithelial cells.

Strains of Yersinia enterocolitica from different O serogroups were tested for their ability to adhere to cultured epithelial cells (H.Ep 2) after growth of bacteria in broth at 22 C or 37 C. Strains that adhered to H.Ep 2 cells generally did so better after growth at 22 C than at 37 C. The data indicate that the ability to produce either of the mannose-resistant (MR) hemagglutinins, MR/Y or K1, each associated with fimbriae, does not correlate with ability to adhere to epithelial cells in this particular in vitro model.

Scanning electron microscopy of L-forms of Yersinia enterocolitica.

An SEM study upon the developmental cycle of Yersinia enterocolitica added further confirmation to its being an L-cycle. It was not found easy to demonstrate flagella by the method, but the conclusion was reached that it has potential as a method for the rapid identification of bacterial L-forms.

Activation of human complement by Yersinia enterocolitica: ultrastructural alterations and C3b-deposition.

Rapid killing of Yersinia enterocolitica strain 75 in smooth form (Ye 75 S) was observed in the presence of serum or of lysozyme-free serum whereas the killing activity of EGTA-serum was slow, and absent in heated (30 min 56 degree C) serum. Similarly, complement (C) activation by Ye 75 S was rapid in serum and lysozyme-free serum but slow via the alternative pathway (EGTA-serum). These data suggest that C is sufficient for killing of the cells and most active via an intact classical pathway. Electronmicroscopic studies were performed on bacterial killed by serum (C + lysozyme) or by lysozyme-free serum (C). In these experiments cell fragmentation and spheroplast formation were seen after exposure of Ye 75 S to serum; in bacteria incubated with lysozyme-free serum "blebs" formation was observed as the most prominent alteration. These blebs most likely originate from the outer membrane as a result of C activation on the cell surface. The deposition of activated C (C3b) on Ye 75 S was analyzed kinetically in the presence of serum or EGTA-serum. With serum (30 vol%) massive C3b deposition was observed within 20--30 min whereas with EGTA-serum (30 vol%) the deposition of C3b was slower and less complete. Experiments with EGTA-serum also revealed that the deposition of C3b started at single sites mainly located in the region of the cell poles; from these sites spreading of C3b occurred until large areas of the cell surface were covered. These data suggest that C activation via the alternative pathway is restricted to certain regions of the bacterial surface.

Yersiniosis in children.

40 cases of bacteriologically proved Yersinia enterocolitica infections in children under 15 years were reviewed. Most children presented with abdominal symptoms, and diarrhoea was present in 80% of them. In half of those with diarrhoea the stools were mucoid and gross blood was often present. Faecal leucocytes were found in 4 out of the 5 children studied. The clinical findings are consistent with the enteroinvasive pathogenic mechanism proposed for Y. enterocolitica. 29 of 30 faecal isolates of Y. enterocolitica were found invasive for human epithelial cells in vitro. Nine strains produced an enterotoxin demonstrable in newborn mouse assay. Toxin production may be an additional pathogenic mechanism in human yersiniosis.

[Sugar composition of the lipopolysaccharide and ultrastructural study of the outer membrane of Yersinia enterocolitica (author's transl)]. / Zuckerzusammensetzung des Lipopolysaccharids und Feinstruktur der äusseren Membran (Zellwand) bei Yersinia enterocolitica.

Phenol-water-extracted lipopolysaccharide (LPS) from Yersinia enterocolitica (strain 75 in smooth form; O-group I), after growth at 10 degrees C, contains approximately 40% (w/w) 6-deoxy-L-altrose. By increasing the cultivation temperature a significant decrease in the O-specific sugar is observed. LPS from cells grown at 40 degrees C contains about 12% 6-deoxy-L-altrose. Thin-section micrographs of cells grown at lower temperatures reveal a distinct layer corresponding to the O-specific side chains of LPS in contrast to cells grown at higher temperatures where a decrease in thickness is observed. It is assumed that this observation is due to the decreasing amount of the O-specific sugar at a higher cultivation temperature. The results obtained by the indirect immunoferritin test indicate that the O-specific polysaccharide layer covers the cell surface completely up to 37 degrees C. However, this layer no longer covers the entire surface of all cells as soon as an incubation temperature of 40 degrees C is applied.