Study of a lipid A-protein complex from Yersinia pseudotuberculosis endotoxin

Mild acid hydrolysis of endotoxin of Yersinia pseudotuberculosis afforded a lipid A--protein complex composed of amino acids and all characteristic components of lipid A: glucosamine, dodecanoic, 3-hydroxytetradecanoic acids and phosphorus in a molar ratio of 2 : 1,5 : 2,8 : 1,7, respectively. The protein component of the complex was shown by gel electrophoresis in the presence of sodium dodecylsulphate to consist of two polypeptides with apparent molecular weights of 12000 and 8000. The lipid A--protein complex cross-reacted with antiserum to endotoxin and lipid A antiserum. The components of the complex, namely lipid A and a protein, are associated tightly but noncovalently and can be separated by ultracentrifugation in the sucrose density gradient after treating the complex with sodium dodecylsulphate. The resultant lipid A and the protein manifest a serological activity.     

Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes.

Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and, furthermore, that YerA is not required to target YopE-Cya proteins to the secretion complex. Taken together, our results suggest that traversing the bacterial and eukaryotic membranes occurs by separate processes that recognize distinct domains of YopE and that these processes are not dependent on YerA activity.     

Isolation and characterization of the immunoglobulin-binding protein from Yersinia pseudotuberculosis

A high molecular weight immunoglobulin-binding protein localized on the surface of bacterial cells has been isolated from the protein fraction of the outer membrane of Yersinia pseudotuberculosis, and its properties are described. The immunoglobulin-binding protein is a trypsin-resistant and temperature-sensitive -structured protein. As shown by MALDI-TOF mass spectrometry, after heating at 100°C the molecular weight of the protein constituted 37.5 kD. The native protein is capable of interacting with human and rabbit IgG but looses the ability to bind the immunoglobulins after the temperature denaturation. The immunoglobulin-binding protein binds to the Fc-fragments of the immunoglobulins and binding depends on the presence of calcium ions.     

Adhesion of Yersinia pseudotuberculosis serotype 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.     

Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.     

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia , the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al ., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK -mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK -mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.     

An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein

Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence-associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein.     

Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty-two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose-binding protein (MBP)--invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl-terminal 192 amino acids retained cell binding activity. One carboxyl-terminal invasin fragment and seven MBP--invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl-terminal 192 amino acids of invasin contains the integrin-binding domain, even though this region does not contain the tripeptide sequence Arg-Gly-Asp.     

A study of the YopD-lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.     

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.     

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.     

Yersinia pseudotuberculosis toxins

The review of publications about protein toxins Y. pseudotuberculosis are presented. It includes the main data obtained by domestic and foreign investigators as well as the results of our own elaboration in the study of Y. pseudotuberculosis protein toxins. The guestions of isolation, purification, characterization of physico-chemical and biological properties, the mechanism action and role of toxins on pathogenesis of infection were discussed.     

Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.     

Expression and insecticidal activity of Yersinia pseudotuberculosis and Photorhabdus luminescens toxin complex proteins

Photorhabdus luminescens toxin complex (Tc) has been characterized as a potent three-component insecticidal protein complex. Homologues of genes encoding P. luminescens Tc components have been identified in several other enterobacteria and in Gram-positive bacteria, showing these genes are widespread in bacteria. In particular, tc gene homologues have been identified in Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis and may have a role in Y. pestis evolution. Y. enterocolitica tc genes have been shown to be active against Manduca sexta larvae. Here, we demonstrate that expression optimization is essential to obtain bioactive P. luminescens Tc proteins and demonstrate that TcaAB and TcdB + TccC are stand-alone toxins against a M. sexta insect model. Moreover, we report that Y. pseudotuberculosis IP32953 Tc proteins are also toxic to M. sexta larvae but do not cross-potentiate as P. luminescens Tc components.     

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague

Yersinia pestis diverged from Yersinia pseudotuberculosis相似文献   

Morphofunctional changes in the chief cells of the intrinsic glands of the stomach in the presence of an excess of glucocorticoid and sex hormones in the body

In 260 male white rats electron microscopically, cytochemically with biochemical estimation of proteolitic activity of gastric juice, it has been demonstrated that principle cells of the gastric glands, after injection of small doses of hydrocorticone, respond with a complex of adequate ultrastructural, cytochemical and functional changes directed to realization of the drugs stimulating effect. With increasing time of the experiment, or the doses, progressively developing distrophic processes, involving predominantly the protein-synthesizing apparatus and, to a less degree, the mitochondrial mechanism, are observed in the principle cells. This results in inhibition of the pepsinogene synthesis phase and in decreasing proteolytic activity of gastric juice. Reaction of the principle cells is independent on testosterone doses; the protein-synthesizing and mitochondrial (to a less degree) apparatuses develop; the pepsinogene extrusion phase is suppressed; it results in its deposition in cytoplasm and in decreasing proteolytic activity of gastric juice. When testosterone and hydrocortisone are injected simultaneously in small doses, the stimulating effect of hydrocortisone to the principle cells is abolished and at large doses it is sharply manifested and is not accompanied with destructive changes in the principle cells.