Biomedical subjects

Guy R Cornelis

Publications and source records attributed to Guy R Cornelis.

At least 19 recordsLinked to original sources

PubMed2006

Escape from immune surveillance by Capnocytophaga canimorsus.

Capnocytophaga canimorsus, a commensal bacterium from dogs' mouths, can cause septicemia or meningitis in humans through bites or scratches. Here, we describe and characterize the inflammatory response of human and mouse macrophages on C. canimorsus infection. Macrophages infected with 10 different strains failed to release tumor necrosis factor (TNF)- alpha and interleukin (IL)-1 alpha . Macrophages infected with live and heat-killed (HK) C. canimorsus 5 (Cc5), a strain isolated from a patient with fatal septicemia, did not release IL-6, IL-8, interferon- gamma , macrophage inflammatory protein-1 beta , and nitric oxide (NO). This absence of a proinflammatory response was characterized by the inability of Toll-like receptor (TLR) 4 to respond to Cc5. Moreover, live but not HK Cc5 blocked the release of TNF- alpha and NO induced by HK Yersinia enterocolitica. In addition, live Cc5 down-regulated the expression of TLR4 and dephosphorylated p38 mitogen-activated protein kinase. These results highlight passive and active mechanisms of immune evasion by C. canimorsus, which may explain its capacity to escape from the host immune system.

Animals↗

PubMed2006

The discovery of SycO highlights a new function for type III secretion effector chaperones.

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

Bacterial Proteins↗

PubMed2006

Length control of extended protein structures in bacteria and bacteriophages.

The length of the tail of bacteriophages is controlled by a protein which acts as a molecular ruler. The needle of the injectisome, which is assembled by the polymerization of subunits that are exported through the nascent injectisome, is functionally related to the tail of bacteriophages. Interestingly, its length is controlled by a protein, which is itself exported and acts as a molecular ruler that is coupled to a substrate specificity switch. The bacterial flagellum is evolutionarily related to the injectisome. The length of the hook is also controlled by a secreted protein. This protein acts as a substrate specificity switch and, possibly, also as a ruler.

Bacteria↗

PubMed2006

The type III secretion injectisome.

The type III secretion injectisome is a complex nanomachine that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. The principal structural components of the injectisome, from the base located in the bacterial cytosol to the tip of the needle protruding from the cell surface, have been investigated in detail. The structures of several constituent proteins were solved at the atomic level and important insights into the assembly process have been gained. However, despite the ongoing concerted efforts of molecular and structural biologists, the role of many of the constituent components of this nanomachine remain unknown.

Bacteria↗

PubMed2005

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles.

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.

Antigens, Bacterial↗

PubMed2005

Type III secretion: the bacteria-eukaryotic cell express.

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future.

Animals↗

PubMed2005

Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes.

Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (wt) P. aeruginosa. wt Y. enterocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN ( Delta HOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of Delta HOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopB/D proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon.

Cell Membrane↗

PubMed2005

Bacterial injectisomes: needle length does matter.

Many pathogenic bacteria use a type III secretion nanomachine (an injectisome) to deliver virulence proteins into the cytosol of their eukaryotic host cells. Most injectisomes possess a stiff needlelike structure of a genetically defined length. We found that a minimal needle length was required for efficient functioning of the Yersinia enterocolitica injectisome. This minimal needle length correlated with the length of the major adhesin at the bacterial surface. The needle may be required for triggering type III secretion, and its length may have evolved to match specific structures at the bacterial and host cell surfaces.

Adhesins, Bacterial↗

PubMed2005

The bacterial injection kit: type III secretion systems.

Type III secretion (T3S) systems are widespread among Gram-negative bacteria pathogenic for animals and plants, including Yersinia spp., Salmonella spp., Shigella spp., enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, or Pseudomonas spp. T3S systems allow bacteria to inject virulence proteins, called T3S effectors, into the cytosol of their eukaryotic host cells. These virulence factors will paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules such as small guanosine triphosphate (GTP)-binding proteins, mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-kappaB, or phosphoinositides. The activity of T3S effectors allows bacteria, for example, to invade non-phagocytic cells or to inhibit phagocytosis, to downregulate or promote pro-inflammatory responses, to induce apoptosis, to prevent autophagy, or to modulate intracellular trafficking. In this review, we present the most recent advances in the understanding of the mode of action of T3S effectors. We highlight the biochemical activities of these eukaryotic-like bacterial proteins that are shared among pathogens carrying T3S systems and the sequence, structural and functional resemblances between T3S effectors and eukaryotic proteins.

Animals↗

PubMed2005

Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT.

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel beta-sheet flanked by three alpha-helices, SycT lacks the dimerization alpha-helix and has an additional beta-strand capable of undergoing a conformational change. The dimer interface consists of two beta-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group.

Amino Acid Sequence↗

PubMed2005

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica.

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C-terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original alpha/beta fold, a P-x-L-G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.

Amino Acid Sequence↗

PubMed2005

Secretion of YscP from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity.

The length of the needle of the Yersinia Ysc injectisome is determined by a protein called YscP. This protein, which acts both as a molecular ruler and as a substrate-specificity switch for type III secretion is itself secreted by the injectisome. In this report, we address the question why YscP is secreted. By a systematic deletion analysis and by fusing different parts of the molecule to the adenylate cyclase reporter, we identified two independent secretion signals. One of them is encompassed within the 35 N-terminal residues while the second one spans residues 97-137. These two signals are functionally different from Yop secretion signals. When both secretion signals were removed, Yops could still be secreted but the needle length control was lost. YscP possessing only one signal did not control needle length properly but the control was improved when more YscP was produced and secreted. YscP deprived of both signals could not control length, even when overproduced. We conclude from this that YscP needs to be secreted to exert its length control function but not its substrate-specificity switch function.

Bacterial Proteins↗

PubMed2005

Type III secretion: more systems than you think.

The type III secretion (T3S) pathway allows bacteria to inject effector proteins into the cytosol of target animal or plant cells. T3S systems evolved into seven families that were distributed among Gram-negative bacteria by horizontal gene transfer. There are probably a few hundred effectors interfering with control and signaling in eukaryotic cells and offering a wealth of new tools to cell biologists.

Animals↗

PubMed2004

Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1-mediated maturation and release of interleukin-1beta.

Yersinia bacteria can take control of the host cell by injecting so-called Yop effector proteins into the cytosol of the cells to which they adhere. Using Yersinia enterocolitica strains that are deficient for one or more Yops, we could show that YopE and, to a lesser extent, YopT interfere with the caspase-1-mediated maturation of prointerleukin-1beta in macrophages. In addition, overexpression of YopE and YopT was shown to prevent the autoproteolytic activation of caspase-1 in a way that is dependent on their inhibitory effect on Rho GTPases. Expression of constitutive-active or dominant-negative Rho GTPase mutants or treatment with Rho GTPase inhibitors confirmed the role of Rho GTPases and, in particular, Rac1 in the autoactivation of caspase-1. Rac1-induced caspase-1 activation was mediated by its effect on LIM kinase-1, which is targeting the actin cytoskeleton. Rac-1 and LIM kinase-1 dominant-negative mutants were shown to inhibit caspase-1 activation induced by overexpression of Asc, which is a caspase-1-activating adaptor protein. Moreover, Rac1 as well as YopE and YopT significantly modulated caspase-1 oligomerization. These results highlight a previously unknown function of Rho GTPases in the activation of caspase-1 and give new insight on the role of YopE in immune-escape mechanisms of Yersinia.

Animals↗

PubMed2004

Identification of a nuclear targeting signal in YopM from Yersinia spp.

YopM is a type III secretion effector from Yersinia which contributes to pathogenicity but whose action still remains unclear. It is an acidic, leucine-rich repeats (LRR) containing protein which migrates to the nucleus of target cells in spite of the fact that it does not contain any classical nuclear localization signal (NLS). Using a yeast approach, we observed that the three first LRRs (LRR1-3) and the 32 C-terminal residues of YopM (YopMC-ter) act as NLSs in yeast. Furthermore, by transfection of HEK293T cells, we observed that YopMC-ter could direct large recombinant EGFP-LexA-AD proteins into the nucleus of mammalian cells confirming that it contains a NLS. Critical residues for nuclear targeting were identified by site-directed mutagenesis in YopMC-ter. In addition, we show that YopMC-ter NLS is crucial for the nuclear targeting of an EGFP-YopM fusion protein.

Active Transport, Cell Nucleus↗

PubMed2004

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica.

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

Bacterial Outer Membrane Proteins↗

PubMed2003

The needle length of bacterial injectisomes is determined by a molecular ruler.

Size determination represents a fundamental requirement for multicomponent biological structures. Some pathogenic bacteria possess a weapon derived from the flagellum. Like the flagellum, this type-III secretion apparatus, called the injectisome, has a transmembrane basal body, but the external component is a needle-like structure instead of a hook and a filament. Here, we provide evidence that the length of this needle is determined by the size of a protein, YscP, acting as a molecular ruler.

Alleles↗

PubMed2003

The multitalented type III chaperones: all you can do with 15 kDa.

Despite the fact that type III chaperones were discovered approximately 10 years ago, the precise role of most of them is still mysterious. A panoply of functions has been proposed for the members of this family of proteins. Type III chaperones have been suggested to act as anti-aggregation and stabilizing factors. They have also been proposed to keep their substrates in unfolded or partially folded structures, set a hierarchy on secretion, and participate in the regulation of the transcription of the type III substrates. Here, we review this enigmatic family of proteins, and discuss the experimental data supporting the roles proposed for type III chaperones.

Bacterial Proteins↗