Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.     

Secretion of Yop proteins by Yersiniae.

Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of the genus Yersinia cease growing and produce large amounts of a series of plasmid-encoded proteins involved in pathogenicity. These proteins, called Yops (for Yersinia outer membrane proteins), are detected in both the outer membrane fraction and the culture supernatant. We present here the nucleotide sequence of genes yop20 and yop25 from Yersinia enterocolitica O:9. Protein Yop25 is very similar to YopE, the corresponding protein from Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica O:8 (A. Forsberg and H. Wolf-Watz, J. Bacteriol. 172:1547-1555, 1990). This is the first report of a yop20 sequence of yersiniae. We present evidences that Yops are not membrane proteins. Their detection in the membrane fraction results either from copurification of large aggregates of extracellular Yops with the membrane fraction or from the adsorption of released proteins to the cell surface. In contrast with Yops, protein P1 has characteristics of a true membrane protein. The release of Yops by Y. enterocolitica occurs by a novel secretion mechanism that does not involve the cleavage of a typical signal sequence or the recognition of a carboxy-terminal domain.     

Expression of antigens encoded by the virulence plasmid of Yersinia enterocolitica under different growth conditions.

The expression of polypeptides of the virulence plasmid of Yersinia enterocolitica serotype O:3 was studied with the immunoblotting technique and specifically absorbed antisera to Y. enterocolitica O:3. At least 16 polypeptides were apparently specified by the virulence plasmid when plasmid-bearing bacterial were grown at 37 degrees C or intraperitoneally in semipermeable capsules. The different growth media used (also with added Ca2+) had quantitatively or qualitatively only a little influence on the expression of these polypeptides, whereas the growth temperature had a stronger influence. The best expression was achieved at 37 degrees C, at 22 degrees C the expression was weak, and at 4 degrees C the plasmid genes were inactive. Two chromosomally encoded polypeptides were expressed only at 37 degrees C, whereas the expression of eight polypeptides expressed at 22 degrees C was repressed at 37 degrees C. The intraperitoneal growth in capsules was used to detect the virulence plasmid-associated polypeptides of Yersinia pestis. Four plasmid-associated polypeptides were detected in Y. pestis with antiserum to Y. enterocolitica virulence plasmid antigens, and three were detected with antiserum to Y. pestis EV76. These results suggested that the virulence plasmid of Y. pestis was activated in the interstitial environment in vivo, where Ca2+ concentration was high, and also that the virulence plasmids of Y. enterocolitica and Y. pestis have three to four immunologically related polypeptides in common.     

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.     

Iron uptake and iron-repressible polypeptides in Yersinia pestis.

Pigmented (Pgm+) cells of Yersinia pestis are virulent, are sensitive to pesticin, adsorb exogenous hemin at 26 degrees C (Hms+), produce iron-repressible outer membrane proteins, and grow at 37 degrees C in iron-deficient media. These traits are lost upon spontaneous deletion of a chromosomal 102-kb pgm locus (Pgm-). Here we demonstrate that an Hms+ but pesticin-resistant (Pst(r)) mutant acquired a 5-bp deletion in the pesticin receptor gene (psn) encoding IrpB to IrpD. Growth and assimilation of iron by Pgm- and Hms+ Pst(r) mutants were markedly inhibited by ferrous chelators at 37 degrees C; inhibition by ferric and ferrous chelators was less effective at 26 degrees C. Iron-deficient growth at 26 degrees C induced iron-regulated outer membrane proteins of 34, 28.5, and 22.5 kDa and periplasmic polypeptides of 33.5 and 30 kDa. These findings provide a basis for understanding the psn-driven system of iron uptake, indicate the existence of at least one additional 26 degrees C-dependent iron assimilation system, and define over 30 iron-repressible proteins in Y. pestis.     

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.     

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.     

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.     

Expression of the temperature-inducible outer membrane proteins of yersiniae.

The expression of the temperature-inducible plasmid-coded outer membrane proteins (YOPs) of Yersinia pseudotuberculosis was studied. These proteins were not recovered in the outer membrane fraction when the strain was grown in minimal medium at 37 degrees C, but they were expressed under these conditions. A strict correlation was found between Ca2+ dependency in the virulent strain, YPIII(pIB1), and ability to express YOPs. Ca2+-independent plasmid mutants or RNA-polymerase mutants harboring the virulence plasmid were unable to express YOPs, in contrast to the wild-type strain. These strains were also found to be avirulent. Sera recovered from patients or animals undergoing infection with either Y. pseudotuberculosis, Y. pestis, or Y. enterocolitica possessed antibodies directed against YOPs, indicating that they were expressed in all three pathogenic Yersinia species during infection. The YOPs of the three different species showed high immunological relatedness.     

Identification and mapping of the temperature-inducible, plasmid-encoded proteins of Yersinia spp.

The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. B?lin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.     

Plasmid-mediated and temperature-regulated surface properties of Yersinia enterocolitica.

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid which codes for a series of novel outer membrane proteins. The expression of these proteins on the outer membrane is temperature regulated: when cells are grown at 25 degrees C, these proteins are not exposed on the outer membrane, whereas they occur in high copy number when cells are grown at 37 degrees C. The majority of these proteins are externally exposed on the cell surface as evidenced by their susceptibility to proteolysis by exogenously added proteases. The expression of the plasmid-mediated proteins on the outer membrane does not favor adherence of the bacteria to intestinal epithelial cells in vitro. Cultures grown at 25 degrees C adhered to Henle cell monolayers, whereas those grown at 37 degrees C did so much less effectively. The presence of the proteins on the bacterial surface appears to be involved in rendering the cells resistant to the bactericidal effects of serum, i.e., 37 degrees C-grown cells were resistant to serum killing, and removal of the outer membrane proteins with pronase rendered them sensitive. Evidence is presented which strongly suggests that the plasmid-mediated proteins are synthesized and expressed on the cell surface either during or after transit of the ingested bacteria to the lamina propria. Some properties afforded to the cells by the outer membrane proteins are described.     

Growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 inhibited on cefsulodin-Irgasan-novobiocin agar.

A total of 169 strains of Yersinia spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan-novobiocin (CIN) agar containing 15 or 4 micrograms of cefsulodin per ml, on salmonella-shigella agar, and on MacConkey agar. CIN media inhibited the growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 (3B/O3) but not the growth of the other Yersinia organisms used. Relative to growth on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 6% yeast extract, 48 and 44% of Y. pseudotuberculosis and Y. enterocolitica 3B/O3 strains, respectively, were inhibited on CIN I agar (low cefsulodin concentration), and 83 and 54%, respectively, were inhibited on CIN II agar (high cefsulodin concentration) after incubation for 24 h at 32 degrees C. The inhibition of Y. pseudotuberculosis growth was significantly more extensive on CIN II agar than on CIN I agar. The MICs of cefsulodin and novobiocin clearly indicated a higher susceptibility for Y. pseudotuberculosis than for the other Yersinia organisms at 32 degrees C. All Y. pseudotuberculosis strains were susceptible to cefsulodin at 15 micrograms/ml (the approximate concentration used in CIN II agar). Y. enterocolitica 3B/O3 strains were resistant to cefsulodin, Irgasan, and novobiocin at the concentrations used in CIN media. These findings show that cefsulodin inhibits the growth of Y. pseudotuberculosis at the concentration used in CIN media and that growth inhibition of Y. enterocolitica 3B/O3 is related to a component of the CIN Base.     

Effects of growth temperature, 47-megadalton plasmid, and calcium deficiency on the outer membrane protein porin and lipopolysaccharide composition of Yersinia pestis EV76.

The expression of several virulence determinants of Yersinia pestis is known to be dependent on the in vitro growth temperature. One of these, calcium dependence, is associated with the presence of a 47-megadalton plasmid. We have examined the effects of incubation temperature, calcium in the growth medium, the presence of the 47-megadalton plasmid on the outer membrane protein, and the lipopolysaccharide composition of Y. pestis EV76. When cells were grown at 37 degrees C as opposed to 26 degrees C, a change in lipopolysaccharide composition and a decrease in the amount of an outer membrane protein (protein E) were observed. The lipopolysaccharide obtained from cells incubated at 37 degrees C had a lower proportion of 2 keto-3-deoxyoctanate, a lower phosphate to 2-keto-3-deoxyoctanate ratio, and an increased gel mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis when compared with lipopolysaccharide obtained from cells grown at 26 degrees C. Because of its growth temperature-related abundance, we investigated the nature of protein E. This protein had physical properties similar to those of other enterobacterial porins, including apparent formation of an oligomer on sodium dodecyl sulfate-polyacrylamide gels when solubilized at low temperature, acidic isoelectric point, and strong noncovalent association with the peptidoglycan. Protein E was purified and shown to form an aqueous channel in planar lipid membranes with a conductance of 1.1 nS in 1 M KCl. In addition to growth temperature-related alterations in the lipopolysaccharide and porin components of the outer membrane, the amount of three spots in two-dimensional polyacrylamide gels was shown to be related to the temperature or the presence of calcium during growth. One of these spots was shown to contain residual unmodified portions of two major heat-modifiable proteins which failed to shift to their heat-modified positions on gels, despite solubilization at 100 degrees C for 10 min before electrophoresis. The other two spots were the heat-modified and unmodified forms of another outer membrane protein (J) which did not appear in the isoelectric focusing gel of cells grown at 37 degrees C. It is proposed that the appearance of these spots in two-dimensional analyses is related to the lipopolysaccharide composition of the cells from which the outer membrane is derived and reflects lipopolysaccharide-protein interactions or calcium-protein interactions.     

Expression of iron-regulated proteins in Yersinia species and their relation to virulence.

Under iron-starvation conditions, the different Yersinia species expressed various iron-regulated proteins. Among them, two high-molecular-weight outer membrane proteins were synthesized in high-virulence-phenotype Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovars O:8 and O:Tacoma but were present neither in low-virulence phenotype Y. enterocolitica serovars O:3 and O:9 nor in avirulent Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. enterocolitica serovar O:39. Thus, the degree of virulence correlates with the presence of the two high-molecular-weight proteins in Yersinia species.     

New genes involved in Yersinia pestis fraction I biosynthesis.

Antigenic and immunochemical properties of Yersinia pestis fraction I (FI) preparations extracted by different methods were studied with polyclonal and monoclonal antibodies. The existence of mature FI in a form of a complex antigen whose subunits have different genetic control was demonstrated. Galactolipid was shown, with caf1 product, to be the second species-specific component of the FI complex molecule and is probably encoded by chromosomal genes. It, like caf1 product, was expressed in higher quantities at 37 degrees C than at 28 degrees C. Among FI subunits there were at least two proteins of 28 +/- 2 kDa and 43 +/- 2 kDa which were not specific for Y. pestis but were found also in all Yersinia spp. and some other bacteria. These proteins were synthesised independently of the incubation temperature (4 degrees-40 degrees C) and are possibly encoded chromosomally but outside the caf operon and galactolipid-encoding genes. Both proteins together with galactolipid comprise an envelope antigen found in pFra- or plasmidless Y. pestis strains. Organisation of Y. pestis FI (mature capsular antigen) in the form of a complex of the envelope antigen and the caf1 product is discussed.     

Differentiation of serological responses to Yersinia enterocolitica serotype O9 and Brucella species by immunoblot or enzyme-linked immunosorbent assay using whole bacteria and Yersinia outer membrane proteins.

Serum samples from 134 patients showing by the microagglutination test serological cross-reactivity between Yersinia enterocolitica serotype O9 and Brucella spp. were analyzed by immunoblot and enzyme-linked immunosorbent assay techniques for the presence of antibodies directed against plasmid-encoded, yersinia-associated outer membrane proteins (OMPs). Since these OMPs are exclusively expressed in pathogenic strains of Yersinia spp., this characteristic was chosen for serological differentiation of infections caused by these bacteria. The presence of antibodies against plasmid-encoded OMPs of pathogenic Yersinia spp. in patient sera appeared to be a suitable means to identify acute or recent infection with Y. enterocolitica serotype O9, whereas the failure to detect such antibodies indicated an acute or recent infection with Brucella spp.     

Characterization of the outer membrane proteins of Bordetella avium.

The outer membrane proteins of Bordetella avium were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarkosyl-insoluble outer membrane protein-enriched profiles from 50 virulent B. avium isolates, containing major 21,000- and 37,000-molecular-weight proteins (21K and 37K proteins, respectively) and at least 13 less intensely stained proteins with molecular weights ranging from 13,500 to 143,000, were very similar. The 21K, 27K, 31K, and 37K outer membrane proteins were shown to be associated noncovalently with the underlying peptidoglycan layer. It was necessary to treat cell envelopes with 2% sodium dodecyl sulfate and at temperatures in excess of 60 degrees C for 15 min to release these proteins. Exposure of proteins on the cell surface of B. avium was assessed by labeling with 125I followed by electrophoresis. As many as 13 bands were present in profiles from labeled whole cells. Of the surface-labeled bands, eight corresponded to bands in a radiolabeled outer membrane preparation. The outer membrane protein profile of B. avium was compared with profiles from other Bordetella spp., including 20 B. avium-like and 16 B. bronchiseptica strains isolated from turkeys. The outer membrane protein profile of B. avium was distinctly different from those of the other bordetella. The effect of variations in the growth medium on the expression of outer membrane proteins of B. avium was examined. Expression of 22K, 26K, 56K, and 73K proteins was decreased or eliminated by addition of 50 mM MgSO4 to the medium.     

Anti-V antigen antibody protects macrophages from Yersinia pestis -induced cell death and promotes phagocytosis

The pathogenic Yersinia spp. harbor a common plasmid (pYV) essential for virulence. The plasmid encodes a type III secretion system that functions to translocate Yersinia outer proteins (Yops) into the host cytosol. Within the host cell, the Yops act to inhibit phagocytosis and induce apoptosis. One of the plasmid-encoded proteins, virulence antigen (V), is a major protective immunogen that is involved in Yop translocation. Yersinia pestis, like the enteric Yersinia spp., was both resistant to phagocytosis by and cytotoxic for J774.A1, a murine macrophage cell line. Both of these activities were dependent on culture of the bacteria at 37 degrees C for 1.5-2 h before infection. However, extending the preculture period at 37 degrees C to 24 h, which induced formation of a capsule, completely blocked cytotoxicity. Treating the bacteria with either rabbit polyclonal anti-V antibodies (R anti-V) or monoclonal antibody (MAb) 7.3, antibodies specific for V and protective against plague in vivo, protected J774.A1 cells from Y. pestis -induced cell death and also reversed the inhibition of phagocytosis. Whereas protection against cell cytotoxicity was afforded by the F(ab')(2) portion of R anti-V, the ability of anti-V to induce uptake of Y. pestis appeared to be dependent on the Fc portion of the Ab. The protective epitope(s) recognized by R anti-V was contained in the central region of Y. pestis V (aa 135-275) and were partially cross reactive with Y. pseudotuberculosis and Y. enterocolitica serotype 08 V antigens.