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.     

Virulence and viability of Yersinia pestis 25 years after lyophilization

When equal volumes of 6% lactose and a broth culture of Yersinia pestis were mixed before freezing, approximately 50% of the cells survived lyophilization and reconstitution on the following day. Concomitantly, the number of viable cells per 50% lethal dose increased from about 16 to 125 organisms. On subsequent storage of the lyophilized cells under vacuum in glass ampoules at 4 degrees C for 25 years, more than 25% of the cells remained viable. When stored cultures were assayed immediately after reconstitution, virulence for mice was significantly reduced (as many as 4,000 cells/50% lethal dose), but the virulence was fully restored when reconstituted cultures were held for 24 h at room temperature, or when a subculture was prepared in fresh medium.     

Study of suspected plague cases for isolation and identification of Yersinia pestis

A total of 62 suspected patients of plague were investigated for evidence of Yersinia pestis, by blood culture, lymph node aspirate culture, sputum culture, animal inoculation and serology for f1 antibodies against f1 antigen of Yersinia pestis. None of the samples was positive by direct smear examination and culture for Yersinia pestis, as well as for serology. The non positivity of the cultures is discussed.     

Elimination of virulence plasmids of Yersinia pseudotuberculosis as affected by fluoroquinolones

Pefloxacin, ciprofloxacin and norfloxacin were shown to induce elimination of the virulence plasmids in the strains of Y. pseudotuberculosis. Pefloxacin had a more pronounced eliminating effect than ciprofloxacin and norfloxacin. The elimination of the virulence plasmids with the molecular weight of 40-50 MD in the strains of Y. pseudotuberculosis was more frequent at the subinhibitory concentrations of the fluoroquinolones or at 1/4 of the fluoroquinolone MIC., At 1/8 of the MIC it was rarer. The virulence plasmid elimination in the strains was observed in spite of their serovar but was more frequent in the serovar 1B strains.     

Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS100, has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis. These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis. The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.     

Failure to detect binding of LcrH to the V antigen of Yersinia pestis

V antigen of Yersinia pestis has been reported to bind the chaperone LcrH. We were unable to demonstrate this interaction. Despite methodological differences between our study and an earlier study, we believe that the previous findings were artifactual. One likely confounding element was the tendency of LcrH to adhere on its own to metal chelation chromatographic resin.     

The deamination of L-arginine and the synthesis of active nitrogen compounds in Yersinia pestis

The investigations of authors concerning infants and their families were generalized on the basis of the observations of infants and their parents with some psychological problems in families. The principles of psychotherapeutic work in above-mentioned groups were formulated. Besides the methods of the diagnosis of psychical deviations development in first year infants as well as peculiarities of alterations in mother-baby system and the main forms of psychic disturbances correction in infants were also described.     

YscO of Yersinia pestis is a mobile core component of the Yop secretion system

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca2+-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degreesC in the absence of Ca2+. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37 degreesC in the presence of Ca2+. In the absence of Ca2+, the mutant grew independently of Ca2+, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37 degreesC irrespective of the presence of Ca2+ and in the culture medium in the absence of Ca2+. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

Comparative characterization of the physico-chemical properties of lipopolysaccharides of Yersinia pestis and R-mutants of enterobacteria]

Electrokinetic potentials (EKP) of the cells of R mutants of Escherichia coli and Salmonella minnesota and cells of Yersinia pestis strains EV (line NIIEG), 358/12 P-, TWJ, Java, and 231 (708) were determined, as well as EKP of lipopolysaccharide (LPS) preparations isolated from these bacteria. The electric characteristics of the cell surfaces of the strains under investigation were demonstrated to correlate with the LPS charge and the reduction extent of their molecules. Acidic hydrolysis of LPS on the cell surface resulted in the leveling of the distinctions in EKP values (their reduction to the same level). EKP values and the size of LPS micelles of the studied Y. pestis strains corresponded to those of the deep R mutants of enterobacteria, while the aggregation extent of the molecules was higher for Y. pestis.     

Expression of plasminogen activator pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix

The effect of the plasminogen activator Pla of Yersinia pestis on the adhesiveness of bacteria to the mammalian extracellular matrix was determined. Y. pestis KIM D27 harbors the 9.5-kb plasmid pPCP1, encoding Pla and pesticin; the strain efficiently adhered to the reconstituted basement membrane preparation Matrigel, to the extracellular matrix prepared from human lung NCI-H292 epithelial cells, as well as to immobilized laminin. The isogenic strain Y. pestis KIM D34 lacking pPCP1 exhibited lower adhesiveness to both matrix preparations and to laminin. Both strains showed weak adherence to type I, IV, and V collagens as well as to human plasma and cellular fibronectin. The Pla-expressing recombinant Escherichia coli LE392(pC4006) exhibited specific adhesiveness to both extracellular matrix preparations as well as to laminin. The Pla-expressing strains showed a low-affinity adherence to another basement membrane component, heparan sulfate proteoglycan, but not to chondroitin sulfate proteoglycan. The degradation of radiolabeled laminin, heparan sulfate proteoglycan, or human lung extracellular matrix by the Pla-expressing recombinant E. coli required the presence of plasminogen, and degradation was inhibited by the plasmin inhibitors aprotinin and alpha2-antiplasmin. Our results indicate a function of Pla in enhancing bacterial adhesion to extracellular matrices. Y. pestis also exhibits a low level of Pla-independent adhesiveness to extracellular matrices.     

Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.     

The pH 6 antigen of Yersinia pestis binds to beta1-linked galactosyl residues in glycosphingolipids

The Yersinia pestis pH 6 antigen was expressed by, and purified from, Escherichia coli containing cloned psa genes. By an enzyme-linked immunosorbence-based assay, purified pH 6 antigen bound to gangliotetraosylceramide (GM1A), gangliotriaosylceramide (GM2A), and lactosylceramide (LC) (designations follow the nomenclature of L. Svennerholm [J. Neurochem. 10:613-623, 1963]). Binding to GM1A, GM2A, and LC was saturable, with 50% maximal binding occurring at 498 +/- 4, 390, and 196 +/- 3 nM, respectively. Thin-layer chromatography (TLC) overlay binding confirmed that purified pH 6 antigen bound to GM1A, GM2A, and LC and also revealed binding to hydroxylated galactosylceramide. Intact E. coli cells which expressed the pH 6 antigen had a specificity similar to that of purified pH 6 in the TLC overlay assay except that nonhydroxylated galactosylceramide was also bound. The binding patterns observed indicate that the presence of beta1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.     

Resistance to lipopolysaccharide mediated by the Yersinia pestis V antigen-polyhistidine fusion peptide: amplification of interleukin-10

We previously showed that injection of homogenous staphylococcal protein A-V antigen fusion peptide into mice delayed allograft rejection and suppressed the major proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) associated with generation of protective granulomas. This study was undertaken to determine if V antigen could prevent endotoxic shock, known to be mediated by excessive production of certain proinflammatory cytokines. After treatment with 50 microg of homogeneous V antigen-polyhistidine fusion peptide (Vh), the 50% lethal dose of purified lipopolysaccharide (LPS) in BALB/c mice immediately rose from 63 microg (normal controls) to 318 microg, fell to near baseline (71 microg) in 6 h, and then slowly rose to a maximum of 566 microg at 48 h before again returning to normal. Injected Vh alone (50 microg) promptly induced the anti-inflammatory cytokine interleukin-10 (IL-10) as well as modest levels of TNF-alpha (an inducer of IL-10) in spleen. Concomitant injection of Vh and an otherwise lethal dose of LPS (200 microg) dramatically decreased levels of TNF-alpha and IFN-gamma in the spleen and peritoneal lavage fluid as compared to values determined for LPS alone. These results would be expected if V antigen directly up-regulated IL-10 that is reported to generally down-regulate proinflammatory cytokines. Mice receiving 200 microg of LPS 48 h after injection of Vh exhibited patterns of cytokine synthesis similar to those observed in endotoxin-tolerant mice, a condition also reported to be mediated by IL-10. These findings suggest that V antigen serves as a virulence factor by amplifying IL-10, thereby repressing proinflammatory cytokines required for expression of cell-mediated immunity.     

DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5

The low-Ca2+-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.     

The mechanism of the inhibiting effect of a Yersinia pestis protein factor on the hormone-stimulated response of human thrombocytes

Thermostable protein factor (20000 Da) was prepared from Yersinia pestis avirulent strain EV 1290. This protein inhibited the aggregation of human platelets as well as elevation of [Ca2+], induced by different agonist. The action of this protein on platelets is accompanied by elevation of cellular cGMP levels. This factor activated guanylate cyclase suspension of human platelets. The data indicate that this protein factor of Y. pestis may possibly release in circulation during decay of bacteria and can change the physiological activity of human blood cells and contribute to the pathogenesis of plague.     

Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore

We have sequenced a region from the pgm locus of Yersinia pestis KIM6+ that confers sensitivity to the bacteriocin pesticin to certain strains of Escherichia coli and Y. pestis. The Y. pestis sequence is 98% identical to the pesticin receptor from Yersinia enterocolitica and is homologous to other TonB-dependent outer membrane proteins. Y. pestis strains with an in-frame deletion in the pesticin receptor gene (psn) were pesticin resistant and no longer expressed a group of iron-regulated outer membrane proteins, IrpB to IrpD. In addition, this strain as well as a Y. pestis strain with a mutation constructed in the gene (irp2) encoding the 190-kDa iron-regulated protein HMWP2 could not grow at 37 degrees C in a defined, iron-deficient medium. However, the irp2 mutant but not the psn mutant could be cross-fed by supernatants from various Yersinia cultures grown under iron-deficient conditions. An analysis of the proteins synthesized by the irp2 mutant suggests that HMWP2 may be indirectly required for maximal expression of the pesticin receptor. HMWP2 likely participates in synthesis of a siderophore which may induce expression of the receptor for pesticin and the siderophore.     

Evaluation of the role of the Yersinia pestis plasminogen activator and other plasmid-encoded factors in temperature-dependent blockage of the flea

Yersinia pestis, the plague bacillus, has a plasminogen activator (pla) gene on the 9.5-kb plasmid pPla that is hypothesized to play a role in producing the foregut blockage in the flea vector that precedes transmission. In this study, however, Y. pestis that lacked pPla, the 70-kb virulence plasmid, or both plasmids, proved able to block Xenopsylla cheopis fleas normally. Blockage rates decreased with increasing environmental temperature for fleas infected with either wild type or pPla- Y. pestis. Thus, procoagulant ability of the Y. pestis pla gene product does not mediate blockage, nor does its ability to induce fibrinolysis at>28 degreesC account for failure to block at elevated temperatures. A Y. pestis strain that lacked all or part of the third plasmid of 110 kb, however, failed to colonize the flea midgut normally, indicating that one or more genes on the large plasmid may be required for vectorborne transmission.