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G R Cornelis

Publications and source records attributed to G R Cornelis.

At least 19 recordsLinked to original sources
PubMed2001

Yersinia enterocolitica YopP-induced apoptosis of macrophages involves the apoptotic signaling cascade upstream of bid.

Yersinia enterocolitica induces apoptosis in macrophages by injecting the plasmid-encoded YopP (YopJ in other Yersinia species). Recently it was reported that YopP/J is a member of an ubiquitin-like protein cysteine protease family and that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF-kappaB pathways. Here we analyzed the YopP/J-induced apoptotic signaling pathway. YopP-mediated cell death could be inhibited by addition of the zVAD caspase inhibitor, but not by DEVD or YVAD. Generation of truncated Bid (tBid) was the first apoptosis-related event that we observed. The subsequent translocation of tBid to the mitochondria induced the release of cytochrome c, leading to the activation of procaspase-9 and the executioner procaspases-3 and -7. Inhibition of the postmitochondrial executioner caspases-3 and -7 did not affect Bid cleavage. Bid cleavage could not be observed in a yopP-deficient Y. enterocolitica strain, showing that this event requires YopP. Disruption of the catalytic core of YopP abolished the rapid generation of tBid, thereby hampering induction of apoptosis by Y. enterocolitica. This finding supports the idea that YopP/J induces apoptosis by directly acting on cell death pathways, rather than being the mere consequence of gene induction inhibition in combination with microbial stimulation of the macrophage.

Amino Acid Sequence↗
PubMed2001

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Animals↗
PubMed2001

Fluorene degradation by Sphingomonas sp. LB126 proceeds through protocatechuic acid: a genetic analysis.

Sphingomonas sp. LB126 is able to utilize fluorene as sole source of carbon and energy. In the present study, a mutagenic vector was constructed and a "plasmid rescue" strategy was set up to isolate a 16.5-kb DNA fragment containing genes required for fluorene degradation. A 14.5-kb portion of the cloned DNA was sequenced revealing thirteen open reading frames. Two encoded hypothetical proteins (FldE and FldY) similar to transcriptional regulators and one (ORF360) located on an IS-like element (ISSsp126) encoded a putative transposase. Three other putative proteins (FldB, FldU and FldV) displayed strong similarity with enzymes of the protocatechuate 4,5-degradation pathway utilized by Sphingomonaspaucimobilis SYK-6 for the degradation of lignin breakdown products. The remaining hypothetical proteins displayed only limited similarity with enzyme sequences available from databases. Suicide plasmid-directed mutagenesis and genetic complementations showed that integrity of the protocatechuate catabolic pathway was an absolute requirement for fluorene degradation to proceed. These findings were further supported by the analysis of metabolites in bacterial culture supernatants obtained from appropriate mutants. The results presented here demonstrated the suitability of the genetic tool constructed and supplied the first genetic evidence for the participation of a protocatechuate 4,5-degradation pathway in a bacterial fluorene degradation pathway.

Bacterial Proteins↗
PubMed2001

Yersinia lead SUMO attack.

Little is known about the mechanism by which Yops, proteins that Yersinia inject into the cytosol of macrophage, cause downregulation of the inflammatory response and diseases such as the plague. Now it appears that Yops are the first bacterial member of a new family of ubiquitin-like proteases.

Animals↗
PubMed2000

Molecular and cell biology aspects of plague.

A 70-kb virulence plasmid (sometimes called pYV) enables Yersinia spp. to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. The Ysc apparatus forms a channel composed of 29 proteins. Of these, 10 have counterparts in almost every type III system. Secretion of some Yops requires the assistance, in the bacterial cytosol, of small individual chaperones called the Syc proteins. These chaperones act as bodyguards or secretion pilots for their partner Yop. Yop proteins fall into two categories. Some are intracellular effectors, whereas the others are "translocators" needed to deliver the effectors across the eukaryotic plasma membrane, into eukaryotic cells. The translocators (YopB, YopD, LcrV) form a pore of 16-23 A in the eukaryotic cell plasma membrane. The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT. YopH is a powerful phosphotyrosine phosphatase playing an antiphagocytic role by dephosphorylating several focal adhesion proteins. YopE and YopT contribute to antiphagocytic effects by inactivating GTPases controlling cytoskeleton dynamics. YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription factor NF-kappaB. It also induces rapid apoptosis of macrophages. Less is known about the role of the phosphoserine kinase YopO/YpkA and YopM.

Animals↗
PubMed2000

Type III secretion: a bacterial device for close combat with cells of their eukaryotic host.

Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, enteropathogenic Escherichia coli and several plant-pathogenic Gram-negative bacteria use a new type of systems called 'type III secretion' to attack their host. These systems are activated by contact with a eukaryotic cell membrane and they allow bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to reach a given compartment and destroy or subvert the target cell. These systems consist of a secretion apparatus made up of about 25 individual proteins and a set of proteins released by this apparatus. Some of these released proteins are 'effectors' that are delivered by extracellular bacteria into the cytosol of the target cell while the others are 'translocators' that help the 'effectors' to cross the membrane of the eukaryotic cell. Most of the 'effectors' act on the cytoskeleton or on intracellular signalling cascades. One of the proteins injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell.

Animals↗
PubMed2000

How to survive in the host: the Yersinia lesson.

The Yop virulon allows Yersinia spp. to resist the immune response of the host by injecting harmful proteins into host cells. It is composed of four elements: (i) type III secretion machinery called Ysc; (ii) a set of proteins required to translocate the effector proteins inside the eukaryotic cells; (iii) a control system, and (iv) six Yop effector proteins.

Animals↗
PubMed2000

YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+.

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.

Bacterial Outer Membrane Proteins↗
PubMed2000

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins.

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

Adhesins, Bacterial↗
PubMed2000

Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE.

A type III secretion-translocation system allows Yersinia adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca(2+)-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant Yersinia devoid of most of the Yop effectors (delta HOPEM and delta THE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE(15)-Cya as efficiently as YopE(130)-Cya. They were also able to deliver YopE(delta 17-77). SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type Yersinia suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca(2+) chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.

Bacterial Outer Membrane Proteins↗
PubMed2000

Assembly and function of type III secretory systems.

Type III secretion systems allow Yersinia spp., Salmonella spp., Shigella spp., Bordetella spp., and Pseudomonas aeruginosa and enteropathogenic Escherichia coli adhering at the surface of a eukaryotic cell to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to destroy or subvert the target cell. These systems consist of a secretion apparatus, made of approximately 25 proteins, and an array of proteins released by this apparatus. Some of these released proteins are "effectors," which are delivered into the cytosol of the target cell, whereas the others are "translocators," which help the effectors to cross the membrane of the eukaryotic cell. Most of the effectors act on the cytoskeleton or on intracellular-signaling cascades. A protein injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. Type III secretion systems also occur in plant pathogens where they are involved both in causing disease in susceptible hosts and in eliciting the so-called hypersensitive response in resistant or nonhost plants. They consist of 15-20 Hrp proteins building a secretion apparatus and two groups of effectors: harpins and avirulence proteins. Harpins are presumably secreted in the extracellular compartment, whereas avirulence proteins are thought to be targeted into plant cells. Although a coherent picture is clearly emerging, basic questions remain to be answered. In particular, little is known about how the type III apparatus fits together to deliver proteins in animal cells. It is even more mysterious for plant cells where a thick wall has to be crossed. In spite of these haunting questions, type III secretion appears as a fascinating trans-kingdom communication device.

Bacterial Outer Membrane Proteins↗
PubMed1999

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

Amino Acid Sequence↗
PubMed1999

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.

Amino Acid Sequence↗
PubMed1999

Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD.

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST-YopB hybrid proteins and SycD in Escherichia coli, we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST-YopB in E. coli. By analysis of a series of truncated GST-YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia. In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB-YopD and LcrV.

Antigens, Bacterial↗
PubMed1999

Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG.

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB-dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5-1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore-forming streptolysin O from Streptococcus pyogenes. Upon infection, cells released the low-molecular-weight marker BCECF (623 Da) but not the high-molecular-weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red-X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG.

Animals↗
PubMed1998

TyeA, a protein involved in control of Yop release and in translocation of Yersinia Yop effectors.

Extracellular Yersinia spp. disarm the immune system by injecting the effector Yersinia outer proteins (Yops) into the target cell. Yop secretion is triggered by contact with eukaryotic cells or by Ca2+ chelation. Two proteins, YopN and LcrG, are known to be involved in Yop-secretion control. Here we describe TyeA, a third protein involved in the control of Yop release. Like YopN, TyeA is localized at the bacterial surface. A tyeA knock-out mutant secreted Yops in the presence of Ca2+ and in the absence of eukaryotic cells. Unlike a yopN null mutant, the tyeA mutant was defective for translocation of YopE and YopH, but not YopM, YopO and YopP, into eukaryotic cells. This is the first observation suggesting that Yop effectors can be divided into two sets for delivery into eukaryotic cells. TyeA was found to interact with the translocator YopD and with residues 242-293 of YopN. In contrast with a yopN null mutant, a yopNDelta248-272 mutant was also unable to translocate YopE and YopH. Our results suggest that TyeA forms part of the translocation-control apparatus together with YopD and YopN, and that the interaction of these proteins is required for selective translocation of Yops inside eukaryotic cells.

Apoptosis↗