Characterization of E-type colicinogenic plasmids from Shigella sonnei.

Colicinogenic plasmids encode toxic proteins which have antagonistic activity against closely related bacteria. This study describes the molecular characterization of three colicinogenic plasmids designated as pSSE3, pSSE and pSSE2, each with a molecular size of ∼6 kb, identified in clinical isolates of Shigella sonnei. Sequence analysis revealed that pSSE and pSSE2 shared extensive sequence homology with each other and with Escherichia coli E-type colicinogenic plasmids. The plasmid pSSE3 lacked an additional gene imparting immunity to colicin E8, a unique feature not observed in any of the previously reported sequences of colicin E3 plasmids. Incomplete digestion of colicinogenic plasmids by restriction endonucleases, metachromatic staining with acridine orange and presence of single stranded initiation (ssi) region confirmed the coexistence of ssDNA along with dsDNA. Plasmid copy number as determined by real-time PCR was found to be about 20. Transmission electron microscopy revealed DNA impairment in test bacteria after colicin exposure. We hypothesize that S. sonnei has acquired E-group colicin plasmids from its close relative E. coli, with their sequences undergoing subtle changes depending on the cohabitation in the same milieu.

Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations.

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.

Acanthamoeba: could it be an environmental host of Shigella?

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998-2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 degrees C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.

Cell wall structures which may be important for successful immunization with Salmonella-Shigella hybrid vaccines.

Three separate lots of S. typhi/S. sonnei hybrid (Ty/Shig) live oral vaccine strain 5076-1C were tested for efficacy in human volunteers challenged with virulent S. sonnei. Two lots (2 and 5) protected volunteers, a third lot (8) did not. The three lots were evaluated by immunological tests and electron microscopy. Lots 2 and 5, which protected, contained bacteria that reacted with anti-flagellar serum and had observable attached flagella and pili. Lot 8, which failed to protect, did not react with anti-flagellar serum, and had no observable pili. There was no correlation between vaccine efficacy and the reaction of IgG in patient's sera in western blot analysis. Surface structures on the Ty-Shig hybrid may be important for generating a protective immune response.

[The plasmid heterogeneity of Shigella sonnei phase-I and -II strains]. / Geterogennost' plazmid shtammov Shigella sonnei I i II faz.

The plasmid composition of S. sonnei standard strains has been studied by the method of electron microscopy of the preparations of plasmid DNA. In S. sonnei cells I-941-HP, phase I, plasmids of 2,500; 5,000; 5,600; 6,100 and 6,800 base pairs, as well as plasmids of 85,000-117,000 and 170,000-235,000 base pairs have been detected. In S. sonnei cells, phase II, plasmids of 2,500; 4,900 and 6,100 base pairs, as well as plasmids of 85,000-109,000 base pairs, have been found. Thus, virulent S. sonnei in phase I contain additional plasmids of 5,600; 6,800; 110,000-117,000 and 170,000-237,000 base pairs. The range of plasmid lengths between 85,000-117,000 and 170,000-237,000 base pairs exceeds the usual background of electron-microscopic studies, which makes it possible to come to the conclusion on the intrastrain heterogeneity of these classes of plasmids. The suggestion has been made that the transition of S. sonnei from phase I to phase II is linked with the loss of fragments of the genetic material, limited by inverted DNA repetitions.

[Dissociation and ultrastructure of the cells of Shigella sonnei isolated from gnotobiotes and obtained in vitro]. / Dissotsiatsiia i ul'trastruktura kletok Shigella sonnei, vydelennykh ot gnotobiontov i poluchennykh in vitro.

The dissociation variants of S. sonnei in phase I, isolated from germ-free rats and obtained in vitro, have been studied. Such dissociation variants have been found to form colonies with classical and atypical morphology. The electronmicroscopic study has revealed that different dissociation variants include small dense cells with the markedly thickened cell wall and pronounced microcapsule and spheroplasts with the damaged cell wall and less pronounced microcapsule. The formation of these cells is supposed to be the way of the adaptation of S. sonnei in the course of the infectious process and linked with changes in their virulence in the population cycle.

[Delayed-type hypersensitivity in mice immunized with ribosomal vaccines against Shigella sonnei]. / Giperchuvstvitel'nost' zamedlennogo tipa u myshei, immunizirovannykh ribosomal'noi vaktsinoi Shigella sonnei.

The capacity of S. sonnei ribosomal vaccine (SRV) for inducing delayed hypersensitivity (DH) was studied in the foot pad test on mice. The test injection of SRV in a dose of 10 micrograms, inducing only transient changes in intact mice, led to a highly pronounced reaction in mice immunized with ribosomes in Freund's complete adjuvant. The mean difference in thickness between the test and control (injected with physiological saline) feet amounted to 0.54 mm on day 16 after immunization in two injections. Immunization in a single injection produced a less pronounced reaction. After the injection of SRV without the adjuvant no DH developed in the animals. Histologically, the reaction was typical for DH in mice: in 24 hours, at the time of maximal swelling, the cell infiltration of the tissues with the prevalence of mononuclear cells and a significant proportion of neutrophils was observed. The specificity of this reaction was confirmed by cross tests in mice immunized with SRV and bovine serum albumin: positive reactions were observed in homologous systems only. The independence of the foot pad reaction to SRV from antibody formation was corroborated by the fact that the peak of humoral response occurred two weeks before the development of cutaneous hyperreactivity. It was also shown that, in contrast to antibody formation, the foot pad reaction was completely resistant to the treatment of mice with cyclophosphamide in a dose of 200 mg/kg.

[Ribosomal dysentery vaccine. Study of response and antigenic activity in healthy volunteers]. / Ribosomal'naia dizenteriinaia vaktsina. Izuchenie reaktogennosti i antigennoi aktivnosti na zdorovykh dobrovol'tsakh.

In earlier studies Shigella sonnei ribosomal vaccine was shown to be highly protective for guinea pigs and monkeys. The object of the present study, carried out in 20 healthy volunteers, was the safety and the antigenic activity of this vaccine. The subcutaneous injection of the ribosomal vaccine in doses of 100 micrograms and 200 micrograms produced no febrile reactions nor biochemical and histological changes. The minimal local reaction was observed after injection into the subscapular region: in this case 200 micrograms of the vaccine produced neither severe, nor moderate reactions. A single injection of this dose led to a more than 4-fold rise in the levels of total and cysteine-resistant O-antibodies, as well as to the prolonged elevation of the complement level in the serum.

[Electron microscopic study of experimental infection. II. A study of the dynamics of Shigella infection]. / Elektronno-mikroskopicheskoe izuchenie éksperimental'noi infektsii. Soobshchenie II. Issledovanie dinamiki shigelleznoi infektsii.

The dynamics of experimental Shigella infection of chick embryo fibroblasts was studied with the use of electron microscopy. The antibiotic-resistant forms of Sh. sonnei 1,188 was found to be incapable of invasion into the fibroblast cytoplasm and intracellular proliferation. The destruction of fibroblasts observed during the infection was seemingly caused by the action of bacterial endotoxins.

Purification and the ultrastructure of a bacteriocin produced from Shigella sonnei strain 100052.

A bacteriocin produced by strain 100052 of Shigella sonnei (shigellacin 52) was purified about 80-fold from a mitomycin C-induced culture supernatant. The purified preparation gave a single band on sodium dodecyl sulfate-gel electrophoresis with an approximate molecular weight of 10,000. The negatively stained electron micrograph of the purified bacteriocin preparation revealed the existence of three different particles. They were a small cylindrical tube (4.2 by 6.0 nm), a ring-shaped particle and a filamentous structure. The molecular weight calculated from the shape of the small tube was approximately 50,000. The latter two particles appeared to be constructed from subunits which were morphologically similar to the cylindrical tube. These results suggested that the morphological subunit of shigellacin 52 consisted of five chemical subunits with molecular weights of 10,000 each, and it assembled into two types of polymers, a ring-shaped particle and a filamentous structure, which seemed to be the main components of the purified shigellacin 52 preparation.

[Morphologic demonstration of adsorption of Sonne shigellae onto intestinal epithelium]. / Morfologicheskoe proiavlenie adsorbtsii shigell Zonne na épitelii kishechnika.

Electron-microscopy studies of the process of absorption of Shigella sonnei on the epithelium of the intestine villi of Syrian hamstes were carried out. In the study there were used 20 animals and 8 stains of Shigellae isolated from patients with acute dysentery. It was shown that in inoculating into an isolated loop of the intestine adhesion of Shigellae to the surface of border cells was accompanied by rearrangement of glycocalyx with subsequent destruction of microvilli in the zone of adsorption. Simultaneously, lysis of adsorbed bacteria occurred. An assumption is put forward that penetration of Shigellae into the epithelium is a multistaged process which is realised in the phase of adsorption not by one bacterium but by the whole pepulation with the help of soluable substances producing a toxic effect.

[Electron microscopic study of the I, II phases and R-forms of Sh. sonnei]. / Elektronnomikroskopicheskoe izuchenis mikrobnykh kletok I, II faz i R-formy Sh. sonnei

Electron microscopic study of the microbial cells of the I, II phases and the R-form was carried out. Intact cells were examined by negative contrasting, and morphological differences of various bacterial phases were shown: cells of the I phase had a relatively smooth surface, bacteria of the II phase had a smooth surface, but many cell wall fragments were split from them; the surface of the R-form cells was coarse, folded, and cell wall fragments were split from the majority of bacteria. Antigenic determinants responsible for phasic specificity in bacteria of the I and II phases were located at some distance from the external membrane of the cell wall; as to the R-form cells--they were localized on the wall.

Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.