Expression of Yersinia pestis antigens encoded by the Ca2+-dependence plasmid

Antigens coded by the Ca2(+)-dependance plasmid were found in the cultural medium, cytoplasm and outer membranes of the three monoplasmid (pCadV) strains of Yersinia pestis with the different basic properties. The presence of 20 mM of Mg2+ at least in the medium is necessary for optimal expression of these proteins. The existence of strain differences in the bacterial cells reaction to temperature, cultivation medium has been demonstrated. No difference in the pCad-dependent proteins was found in Yersinia pestis and the causative agents of pseudotuberculosis, enterocolitis.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Identification of outer membrane proteins of Yersinia pestis through biotinylation

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with biotin. Tagged proteins were visualised through streptavidin probing of Western blots. Seven biotinylated proteins of Y. pestis were identified including two porins and the putative virulence factor catalase peroxidase.     

Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Connection of outer membrane protein composition in Yersinia pestis cells with intrinsic plasmids]

A set of isogenic derivatives of Yersinia pestis EV strain was obtained including the variants harbouring the different compositions of Yersinia own plasmids. The protein profiles of outer membranes of the set of strains were defined. The polyacrylamide gel electrophoresis has shown the small 6.1 Md plasmid to code an outer membrane protein with mol mass 29 kDa, different from pesticin I, while the heavy 60.0 Md plasmid encodes the 15-16 kDa polypeptide different from monomers of F1 and T-antigens of plague microbe.     

Transport of Ca2+ by Yersinia pestis.

Low-calcium-response, or Lcr, plasmids of yersiniae are known to promote an in vitro nutritional requirement for 2.5 mM Ca2+ at 37 degrees C which, if not fulfilled, results in cessation of growth with induction of virulence functions (Lcr+). The mechanism whereby Ca2+ regulates this metabolic shift is unknown. Radioactive Ca2+ was not actively accumulated by yersiniae but was excluded by an exit reaction analogous to those described for other bacteria. Nevertheless, cultivation at 37 degrees C with 0.1 mM Ca2+, a level insufficient to prevent restriction of cell division, promoted significantly more binding of the cation by Lcr+ organisms than by plasmid-deficient Lcr- mutants. According, Lcr+ yersiniae may possess unique ligands capable of recognizing Ca2+.     

Components of the outer membrane of Yersinia pseudotuberculosis and their role in the pathogenesis of pseudotuberculosis

The pore-forming protein, lipopolysaccharide-protein complex and lipopolysaccharide of Y. pseudotuberculosis outer membrane have been shown to participate in the penetration of the bacteria into the cells of the macroorganism, to produce a toxic effect on these cells and to enhance the ingesting activity of macrophages in small doses, while suppressing it in large doses. When introduced parenterally, protein induces a more pronounced clinical picture of specific reactive hepatitis in experimental animals and greater changes in their kidneys than the lipopolysaccharide--protein complex.     

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.     

Cloning of the locus encoding synthesis of an outer membrane protein (Omp2) of the calcium dependence plasmid of Yersinia pestis (Lehmann, Neumann)

The genetic locus of Yersinia pestis encoding synthesis of a 46-kDa heat-inducible outer membrane protein (Omp2) was cloned into pBR322 plasmid. The Omp2 was shown to be analogous to previously described YopH and Yop2b proteins. The fifth HindIII fragment of 48-MDa calcium dependence plasmid pCad358 mediates production of 31- and 28-kDa proteins, irrespective of orientation of the insertion. A 31-kDa polypeptide seems to correspond to the YopJ described elsewhere. The maps of BamHI and HindIII of pCad358 region studied differed from those described for pCD1 plasmid of Y. pestis KIM. The products encoded by genes from the fragment cloned in the Pgm+ background give rise to considerable growth of Y. pestis within mouse peritoneal macrophages but were not sufficient to cause lethal infectious process.     

DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

A C-terminal region of Yersinia pestis YscD binds the outer membrane secretin YscC

YscD is an essential component of the plasmid pCD1-encoded type III secretion system (T3SS) of Yersinia pestis. YscD has a single transmembrane (TM) domain that connects a small N-terminal cytoplasmic region (residues 1 to 121) to a larger periplasmic region (residues 143 to 419). Deletion analyses established that both the N-terminal cytoplasmic region and the C-terminal periplasmic region are required for YscD function. Smaller targeted deletions demonstrated that a predicted cytoplasmic forkhead-associated (FHA) domain is also required to assemble a functional T3SS; in contrast, a predicted periplasmic phospholipid binding (BON) domain and a putative periplasmic "ring-building motif" domain of YscD could be deleted with no significant effect on the T3S process. Although deletion of the putative "ring-building motif" domain did not disrupt T3S activity per se, the calcium-dependent regulation of the T3S apparatus was affected. The extreme C-terminal region of YscD (residues 354 to 419) was essential for secretion activity and had a strong dominant-negative effect on the T3S process when exported to the periplasm of the wild-type parent strain. Coimmunoprecipitation studies demonstrated that this region of YscD mediates the interaction of YscD with the outer membrane YscC secretin complex. Finally, replacement of the YscD TM domain with a TM domain of dissimilar sequence had no effect on the T3S process, indicating that the TM domain has no sequence-specific function in the assembly or function of the T3SS.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Temperature and Ca(2+)-dependence of the sarcoplasmic reticulum Ca(2+)-ATPase in haddock,salmon, rainbow trout and zebra cichlid

Temperature dependence of Ca(2+)-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degrees C. Whether the break point arises as a result of temperature dependent changes in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca(2+)-dependence of SR Ca(2+)-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degrees C, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the temperature area, 6-14 degrees C. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca(2+)-ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degrees C in salmon and rainbow trout, respectively. Ca(2+)-dependence in the four different fish species investigated was very similar with half maximal activation (K(0.5)) between 0.2 and 0.6 micro M and half maximal inhibition (I(0.5)) between 60 and 250 micro M. Results indicated that interaction between SR Ca(2+)-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated.