Inject Effector Proteins Research Articles

Cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity

Phytopathogenic bacteria use the type-III secretion system (TTSS) to inject effector proteins into plant cells, presumably to colonize their hosts. The function of these proteins inside plant cells has remained a mystery for years. The recent discovery that the effectors XopD, AvrXv4, AvrPphB, and AvrRpt2 have cysteine protease functions reveals that the proteolysis of host substrates is an important strategy employed by pathogens to alter plant physiology. Moreover, the characterization of these proteases and their targets provides new insight to mechanisms of bacterial virulence and the activation of plant immunity.

Xanthomonas type III effector XopD targets SUMO-conjugated proteins in planta.

Xanthomonas campestris pathovar vesicatoria (Xcv) uses the type III secretion system (TTSS) to inject effector proteins into cells of Solanaceous plants during pathogenesis. A number of Xcv TTSS effectors have been identified; however, their function in planta remains elusive. Here, we provide direct evidence for a functional role for a phytopathogenic bacterial TTSS effector in planta by demonstrating that the Xcv effector XopD encodes an active cysteine protease with plant-specific SUMO substrate specificity. XopD is injected into plant cells by the TTSS during Xcv pathogenesis, translocated to subnuclear foci and hydrolyses SUMO-conjugated proteins in vivo. Our studies suggest that XopD mimics endogenous plant SUMO isopeptidases to interfere with the regulation of host proteins during Xcv infection.

Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage

Pathogenic Yersinia species ( Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) make use of a virulence plasmid-encoded type three secretion system (TTSS) to inject effector proteins into host cells. Y. enterocolitica YscM1 (LcrQ in Y. pestis and Y. pseudotuberculosis) and its homologue YscM2 are regulatory components of the TTSS that are also secreted by this transport apparatus. YscM1 and YscM2 share 57% identity and are believed to be functionally equivalent. We have recombinantly expressed and purified YscM1 and YscM2 in Escherichia coli. After expression as glutathione S-transferase (GST) fusions purification to near homogeneity was achieved by glutathione–Sepharose affinity chromatography followed by PreScission protease treatment to cleave off GST and gel filtration on a Superdex 75 column. Such recombinant YscM1 and YscM2 bound efficiently to the specific chaperone SycH, indicating proper folding of the purified proteins. Gel filtration analyses revealed that both YscM1 and YscM2 formed homodimers. The YscM1 and YscM2 homodimers could be dissociated at high ionic strength, indicating that salt bridges essentially contribute to the dimerization. We further demonstrated that YscM1 and YscM2 are susceptible to thrombin cleavage.

Coordinate Regulation of Bacterial Virulence Genes by a Novel Adenylate Cyclase-Dependent Signaling Pathway

Type III secretion systems (TTSSs) are utilized by numerous bacterial pathogens to inject effector proteins directly into host cells. Using a whole-genome microarray, we investigated the conditions and regulatory factors that control the expression of the Pseudomonas aeruginosa TTSS. The transcriptional response of known TTSS genes indicates a hierarchical pattern of expression in which a set of secretion apparatus and regulatory genes is constitutively expressed. Further analysis of genes coordinately regulated with those encoding the TTSS led to the identification of a signaling pathway that originates from a membrane-associated adenylate cyclase and controls TTSS gene expression. Transcriptome analysis of mutants lacking the ability to synthesize cAMP or the cAMP binding protein Vfr implicated this pathway in the global regulation of host-directed virulence determinants, including the TTSS.

Enteropathogenic Escherichia coli translocate Tir and form an intimin-Tir intimate attachment to red blood cell membranes.

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC) the type III secreted protein, Tir, is translocated to the host-cell plasma membrane where it functions as a receptor for the bacterial adhesin intimin, leading to intimate bacterial attachment and "attaching and effacing" (A/E) lesion formation. To study EPEC type III secretion the interaction of EPEC with monolayers of red blood cells (RBCs) has been exploited and in a recent study [Shaw, R. K., Daniell, S., Ebel, F., Frankel, G. & Knutton, S. (2001 ). Cell Microbiol 3, 213-222] it was shown that EPEC induced haemolysis of RBCs and translocation of EspD, a putative pore-forming type III secreted protein in the RBC membrane. Here it is demonstrated that EPEC are able to translocate and correctly insert Tir into the RBC membrane and produce an intimin-Tir intimate bacterial attachment, identical to that seen in A/E lesions. Following translocation Tir did not undergo any change in apparent molecular mass or become tyrosine-phosphorylated and there was no focusing of RBC cytoskeletal actin beneath intimately adherent bacteria, and no pedestal formation. This study, employing an RBC model of infection, has demonstrated that Tir translocation can be separated from host-cell-mediated Tir modifications; the data show that the EPEC type III protein translocation apparatus is sufficient to deliver and correctly insert Tir into host-cell membranes independent of eukaryotic cell functions.

The filamentous type III secretion translocon of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

MRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae.

Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells. The basis for selection of the proteins to be translocated via type III systems is still enigmatic. No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings. We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv. tomato DC3000, to the first 15 codons. Transcription of hrpA starts at a single site 42 bp upstream of the first codon. Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript. These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene. The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

THREADING A NARROW NEEDLE

A HIGH-RESOLUTION CRYSTAL structure obtained by Jorge E. Galan, a microbiologist at Yale School of Medicine, and former postdoc C. Erec Stebbins, now at Rockefeller University, sheds some light on the type III secretion system used by many pathogenic bacteria to inject effector proteins into host eukaryotic cells. structure could help in identifying or designing compounds to interfere with the secretion system. This type III delivery system consists of a needlelike appendage on the surface of the bacterium. bacterial proteins have to travel through this needle to reach the cell. However, the opening of the needle is only about A—too small for a folded protein to fit through. The $64,000 question has always been, 'How do the proteins travel through these particular structures?' Galân says. Each protein is associated with a chaperone protein inside the bacterial cell. chaperones are vital to the operation of the secretion system, but what hasn't been ...

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli.

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3-5 nm. The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55-65 nm and to be raised 15-20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to proposed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution.

Yersinia pestis, the causative agent of bubonic plague, injects effector proteins into the cytosol of mammalian cells that enable the bacterium to evade the immune response of the infected organism by interfering with eukaryotic signal transduction pathways. YopH is a modular effector composed of a C-terminal protein tyrosine phosphatase (PTPase) domain and a multifunctional N-terminal domain that not only orchestrates the secretion and translocation of YopH into eukaryotic cells but also binds tyrosine-phosphorylated target proteins to mediate substrate recognition. The crystal structure of the N-terminal domain of YopH (YopH(N); residues 1-130) has been determined at 2.0 A resolution. The amino-acid sequences that target YopH for secretion from the bacterium and translocation into eukaryotic cells form integral parts of this compactly folded domain. The structure of YopH(N) bears no resemblance to eukaryotic phosphotyrosine-binding domains, nor is it reminiscent of any known fold. Residues that have been implicated in phosphotyrosine-dependent protein binding are clustered together on one face of YopH(N), but the structure does not suggest a mechanism for protein-phosphotyrosine recognition.

YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells.

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.

EspA filament-mediated protein translocation into red blood cells.

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro. As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria-RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria-RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.

Cytolysin-Mediated Translocation (CMT): A Functional Equivalent of Type III Secretion in Gram-Positive Bacteria

Type III secretion for injection of effector proteins into host cells has not been described for Gram-positive bacteria despite their importance in disease. Here, we describe an injection pathway for the Gram-positive pathogen Streptococcus pyogenes that utilizes streptolysin O (SLO), a cholesterol-dependent cytolysin. The data support a model in which an effector is translocated through the SLO pore by a polarized process. The effector, SPN (S . p yogenes N AD-glycohydrolase), is capable of producing the potent second messenger cyclic ADP-ribose, and SLO and SPN act synergistically to trigger cytotoxicity. These data provide a novel paradigm for the function of the cholesterol-dependent cytolysin family and its wide distribution suggests that cytolysin-mediated translocation (CMT) may be the equivalent of type III secretion for Gram-positive pathogens.

The invasion-associated type III secretion system of Salmonella typhimurium: common and unique features.

Several bacterial pathogens make use of a specialized protein secretion system to inject effector proteins into host cells. This system, commonly referred to as type III secretion, is always associated with phenotypes related to intimate interactions between the pathogen and its respective host cells. The enteric pathogen Salmonella typhimurium utilizes a type III secretion system to invade nonphagocytic intestinal epithelial cells. Whereas the invasion-associated type III system of S. typhimurium has evolved to perform a specific function, many of the components of this system are conserved among the type III systems of other bacterial pathogens. This review will discuss the common and unique features of the S. typhimurium system in relation to the type III systems of other human pathogens. Topics discussed include the phenotypes associated with various type III systems, the genetic loci encoding these systems, the components of the type III secretion apparatus, the effector proteins and the mechanisms by which they enter host cells as well as the mechanisms used to regulate the expression of type III systems.

The Yersinia Yop virulon, a bacterial system to subvert cells of the primary host defense.

The Yop virulon enables yersinias (Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It is an integrated system allowing extracellular bacteria to communicate with the host cell's cytosol by injection of effector proteins. It is composed of four elements: (1) a contact or type III secretion system called Ysc, devoted to the secretion of Yop proteins. This secretion apparatus, made of some 22 proteins, recognizes the Yops by a short N-terminal signal that is not cleaved off during secretion; (2) a system designed to deliver bacterial proteins into eukaryotic target cells. This system is made of YopB, YopD and LcrV; (3) a control element (YopN) and (4) a set of effector Yop proteins designed to disarm these cells or disrupt their communications (YopE, YopH, YopM, YpkA/YopO, YopP). The whole virulon is encoded by a 70-kb plasmid called pYV. Transcription of the genes is controlled both by temperature and by contact with a eukaryotic cell.

The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells.

The Yop virulon enables Yersinia spp. (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It is an integrated system allowing extracellular bacteria to communicate with the host cell's cytosol by the injection of effector proteins. It is composed of the following four elements. (I) A contact or type III secretion system called Ysc, which is devoted to the secretion of Yop proteins. This secretion apparatus, comprising some 22 proteins recognizes the Yops by a short N-terminal signal that is not cleaved off during secretion. (II) A system designed to deliver bacterial proteins into eukaryotic target cells. This system is made of YopB, YopD and possibly other Yops such as LcrV. (III) A control element (YopN). (IV) A set of effector Yop proteins designed to disarm these cells or disrupt their communications (YopE, YopH, YpkA/YopO, and YopM). The whole virulon is encoded by a 70 kb plasmid designated pYV. Transcription of the genes is controlled both by temperature and by contact with a eukaryotic cell.