Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Yersinia outer protein P inhibits CD8 T cell priming in the mouse infection model

Pathogenic yersiniae translocate a mixture of effector proteins called Yersinia outer proteins (Yops) into the cytosol of eukaryotic cells by their type III secretion system. YopP is one of the best characterized of these effector proteins and known to inhibit the proinflammatory response of the host by interfering with NF-kappaB signal transduction and inducing apoptosis of macrophages. The effects of YopP on the immune response were studied by a Yersinia Ag-independent approach using bacteria that translocate the well-characterized model Ag listeriolysin O of Listeria monocytogenes via their type III secretion system. In this study we demonstrate a novel function for YopP in vivo. It is shown for the first time that YopP not only counteracts the innate immune defense but also inhibits the adaptive immune system by suppressing the development of an effective CD8 T cell response in a mouse model. A possible mechanism for this could be the inhibition of Ag presentation by dendritic cells (DC). In vitro this is shown to be due to the rapid induction of programmed DC death and to inhibition of DC maturation. Using this approach we could further show that the listeriolysin O-specific CD8 T cells generated in vivo by the yopP mutant are functional and are able to protect mice against a lethal challenge with wild type Listeria.     

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin.

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.     

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.     

Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments.

Soluble proteins can be extracted by osmotic shock of purified rod (photoreceptor cell) outer segments that have intact plasma membranes. The soluble proteins include a component that contains tightly bound GDP-Exchange of this GDP with exogenous nucleotide is catalyzed by (and requires) the membranes from the outer segments. ATP does not participate in these reactions. Approximately one-half of the binding sites in the soluble component require GTP as the source of exogenous nucleotide; the remainder accept GTP or GDP with equal facility. When exogenous GTP is the source of bound nucleotide, it is found in the complex in the form of GDP. Exchange of bound nucleotide with GTP is stoichiometrically related to GTPase activity; this activity is highly dependent upon the presence of both membranes and soluble protein. The soluble nucleotide binding protein was purified by making use of the fact that it binds tightly to the membranes (under conditions of moderate ionic strength) in the absence of GTP and can be eluted by solutions containing low concentrations of GTP (but not GDP or ATP, nor can it be eluted by GTP-free solutions of low ionic strength). The purified protein contains two polypeptide chains of molecular weights 41,000 and 37,000; these are the major species that can be extracted from the outer segments by osmotic shock, and they constitute approximately 7% of the total protein of the isolated organelle.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

Distribution and interrelationship of ubiquitin proteasome pathway component activities and ubiquitin pools in various porcine tissues

The ubiquitin-proteasome pathway fulfills major biological functions, but its physiologic tissue distribution and the interrelationship between pathway component activities and ubiquitin pools are unknown. Therefore, we analyzed free and conjugated ubiquitin, ubiquitin-protein ligation rates (UbPL) and chymotryptic- and tryptic-like proteasome peptidase activities in porcine skeletal muscle, heart, lung, liver, spleen and kidney (n=5 each). There were considerable differences between tissues (p<0.05 for all parameters). Lung and spleen showed high levels of free and conjugated ubiquitin and high UbPL. Proteasome activities were highest in kidney and heart. There were linear relationships between tryptic-like and chymotryptic-like proteasome peptidase activities (r(2) = 0.624, p<0.001) and between free and conjugated ubiquitin tissue levels (r(2) = 0.623, p<0.001). Tissue levels of free and conjugated ubiquitin correlated linear with UbPL (p<0.005), but they were not correlated with proteasome peptidase activities. The results suggest that tissue ubiquitin pools are tightly regulated and indicate a constant proportion of conjugated ubiquitin. They further support the hypothesis that ubiquitin-protein ligase systems, and probably deubiquitylating enzymes, are key regulators of ubiquitin homeostasis. The detected differences are suggestive of tissue-specific roles of ubiquitin-proteasome pathway components. Besides the known importance of the ubiquitin proteasome pathway in heart, kidney and the immune system, the results suggest the lung as another organ in which ubiquitin proteasome pathway components may also significantly contribute to disease processes.     

Small-molecule CB002 restores p53 pathway signaling and represses colorectal cancer cell growth

Much effort is currently focused on the p53 pathway. p53 is a key tumor suppressor, which is mutated or lost in many human cancers. Restoration of the p53 pathway holds the potential to induce selective cell death in tumor cells without harming normal cells that have intact p53 pathways. Most tumor cells express mutated p53 or suppress p53 by overexpression of MDM2. In this study, a compound referred to as CB002 with one closely related compound from the Chembridge library were evaluated for tumor cytotoxicity without affecting normal cells by restoration of the p53 pathway. A decrease of mutant p53 protein expression, restoration of inactivated p53, or some activation of p73 are candidate mechanisms this agent could cause tumor cell apoptosis and growth arrest. We further show that CB002 activates p53 pathway signaling in part via p73 in p53 mutant cancer cell lines. However, it is important to note that we have not established a role for p73 in the anti-tumor effect of CB002 or R1. CB002 causes tumor cell death with synergistic effects with traditional chemotherapeutics CPT-11 and 5-FU.     

The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome

Familial prion diseases are thought to result from a change in structure of the mutant prion protein (PrP), which takes a pathogenic conformation. We have examined the role of molecular chaperones in the folding of normal and mutant PrP Q217R (PrP(217)) in transfected neuroblastoma cells. In a previous report we showed that, although most of the PrP(217) forms escape the endoplasmic reticulum quality control system and aggregate in post-Golgi compartments, a significant proportion of PrP(217) retains the C-terminal glycosylphosphatidyl inositol signal peptide (PrP32), and does not exit the endoplasmic reticulum (Singh, N., Zanusso, G., Chen, S. G., Fujioka, H., Richardson, S., Gambetti, P., and Petersen, R. B. (1997) J. Biol. Chem. 272, 28461-28470). We have now studied the folding and turnover of PrP32 to understand the mechanism by which abnormal PrP forms cause cellular toxicity in our cell culture model and in the human brain carrying the Gerstmann-Str?ussler-Scheinker disease Q217R mutation. In this report, we show that PrP32 remains associated with the chaperone BiP for an abnormally prolonged period of time and is degraded by the proteasomal pathway. This study is the first demonstration that BiP is chaperoning the folding of PrP and plays a role in maintaining the quality control in the PrP maturation pathway. Our data provide new insight into the diverse pathways of mutant PrP metabolism and neurotoxicity.     

The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins ( Yersinia outer proteins [Yops]), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell resulting in cell rounding. YopT is a cysteine protease that cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to inactivation. Small GTPases are modified by isoprenylation of the cysteine of the CAAX box, cleavage of the -AAX tripeptide, and methylation of the cysteine. We have shown that isoprenylation and the endoproteolytic cleavage of the tripeptide of Rho GTPases are essential for YopT-induced cleavage, whereas carboxyl methylation is not required. In the present study, we post-translationally modified RhoA, Rac, Cdc42, and several mutants in vitro and characterized the YopT-induced cleavage with recombinant YopT. We show that farnesylated RhoA is a preferred substrate of YopT compared with the geranylgeranylated GTPase. Geranylgeranylated RhoA, however, is the preferred substrate for YopT-catalyzed cleavage with a threefold faster turnover rate over Rac and Cdc42. Moreover, our data indicate that the composition of the polybasic region of the GTPases defines the specificity and efficiency of the YopT-induced cleavage, and that a space between the polybasic stretch of amino acids at the C terminus and the CAAX box enhances the turnover rate of YopT-catalyzed cleavage.     

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-beta-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST-YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6-p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1.