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PubMed2005

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.

Bacterial Proteins↗

PubMed2003

TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expression and is required for polarized translocation of Yop effectors.

Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.

Bacterial Outer Membrane Proteins↗

PubMed2005

Functional properties of rab15 effector protein in endocytic recycling.

Receptor recycling has emerged as an important regulatory mechanism for cell surface composition, pathogen invasion, and for control over the intensity and duration of receptor signaling in multiple cell types. In the case of the transferrin receptor, receptor recycling is an important step for facilitating iron uptake into the cell, by regulating the availability of the receptor at the cell surface. Following internalization into clathrin-coated pits, the transferrin receptor first enters peripheral sorting endosomes. Here, internalized transferrin receptor is either sorted for recycling back to the cell surface directly, or targeted to a slower route of recycling through a perinuclear population of endosomes termed the endocytic recycling compartment. This chapter describes methodologies to examine the fast and slow modes of transferrin receptor recycling, with a particular emphasis on the function of the novel protein Rab15 effector protein.

Biotin↗

PubMed2000

Role of nonspecific cytotoxic cells in the induction of programmed cell death of pathogenic protozoans: participation of the Fas ligand-Fas receptor system.

Numerous different species of parasites and pathogenic microorganisms produce programmed cell death (PCD) and apoptosis in eukaryotic targets. How ever, only a few studies have demonstrated that effector cells, cytokines, growth factors, or soluble apoptosis-inducing factors are capable of initiating apoptosis in protozoan parasites. Certain Tetrahymena spp. in teleosts are opportunistic pathogens. In the present study these pathogenic protozoans were developed as a model system to describe the potential role of the Fas ligand (FasL)-Fas receptor (FasR) system as a means of innate immunity in teleosts. Nonspecific cytotoxic cells (NCC) constitutively express soluble FasL (sFasL). Binding of the antigen receptor (i.e., NCCRP-1) on NCC to target cells caused the release of sFasL into the milieu. The presence of functional sFasL in these supernatants was determined by Western blot analysis and by demonstrating the lysis of FasR(+) HL-60 but not IM-9 (FasR(-)) targets. Soluble FasL containing supernatants generated by tumor cell-activated NCC also produced a reduction in 2 N DNA (i.e., DNA hypoploidy) of T. furgasoni. The induction of DNA hypoploidy by NCC supernatants could be neutralized by adsorption of the supernatants with anti-FasL antibody (but not with an isotype control). Experiments were next done to determine the expression of FasR on Tetrahymena and study the effects of anti-FasR monoclonal crosslinkage and treatment with soluble human recombinant FasL (huFasL) on initiation of PCD in Tetrahymena. Cell cycle analysis revealed that both crosslinkage and soluble huFasL binding to Tetrahymena produced DNA hypoploidy. The reduction in diploid DNA was confirmed by observing oligonucleosome fragmentation (DNA laddering) following anti-FasR treatment. Additional evidence for FasR expression on Tetrahymena was obtained using fluorescence microscopy and flow cytometry. Both methods showed that all Tetrahymena examined (three species consisting of four isolates) expressed membrane FasR. These studies demonstrated the potential of the FasL-FasR system in teleosts for initiation of antiparasite innate immunity. Effector NCC may initiate PCD of Tetrahymena that express a FasR-like protein. Induction of apoptosis may be a major mechanism of homeostatic control of protozoan parasite infestations/infections.

Animals↗

PubMed2006

Neutrophil influx during non-typhoidal salmonellosis: who is in the driver's seat?

A massive neutrophil influx in the intestine is the histopathological hallmark of Salmonella enterica serovar Typhimurium-induced enterocolitis in humans. Two major hypotheses on the mechanism leading to neutrophil infiltration in the intestinal mucosa have emerged. One hypothesis suggests that S. enterica serovar Typhimurium takes an active role in triggering this host response by injecting proteins, termed effectors, into the host cell cytosol which induce a proinflammatory gene expression profile in the intestinal epithelium. The second hypothesis suggests a more passive role for the pathogen by proposing that bacterial invasion stimulates the innate pathways of inflammation because the pathogen-associated molecular patterns of S. enterica serovar Typhimurium are recognized by pathogen recognition receptors on cells in the lamina propria. A review of the current literature reveals that, while pathogen recognition receptors are clearly involved in eliciting neutrophil influx during S. enterica serovar Typhimurium infection, a direct contribution of effectors in triggering proinflammatory host cell responses cannot currently be ruled out.

Enterocolitis↗

PubMed2003

Clinical response to alefacept: results of a phase 3 study of intramuscular administration of alefacept in patients with chronic plaque psoriasis.

BACKGROUND: Recognition of psoriasis as a T-cell-mediated immune disease has led to the development of various therapeutic approaches directed against the pathogenic T cells. Alefacept, a novel and selective biological recombinant protein, binds CD2 on T cells to block T-cell activation and proliferation and interacts with FcgammaRIII receptors on accessory cells to produce selective T-cell apoptosis. OBJECTIVE: To demonstrate the efficacy and safety of once-weekly alefacept IM compared with placebo given for 12 weeks to patients with chronic plaque psoriasis. METHODS: Multicentre (63 sites in Europe, the USA, and Canada), randomized, double-blind, parallel-group study comparing 10 mg and 15 mg of alefacept and placebo administered IM once weekly for 12 weeks. Patients were followed for an additional 12 weeks after cessation of therapy. Patients were eligible for enrollment if they were > or = 18 years of age, had chronic plaque psoriasis for > or = 12 months involving > or = 10% body surface area, and had CD4+ T-cell counts at or above the lower limit of normal. RESULTS: 507 patients were enrolled into three treatment groups, which were well balanced for demographic, baseline disease characteristics, and treatment history. A significantly greater percentage of patients treated with alefacept 15 mg IM achieved > or = 75% PASI reduction from baseline 2 weeks after the last dose compared with placebo (21% vs. 5%, P < 0.001); 12% of patients treated with alefacept 10 mg IM reached this level of improvement (P = NS vs. placebo). Alefacept was well tolerated, with adverse events similar to that of placebo. CONCLUSIONS: Treatment with alefacept 15 mg IM provided highly significant improvements in all measures of psoriasis disease activity compared with placebo. Pharmacodynamic data confirmed that alefacept is a selective biological agent that reduces memory-effector T cells (CD4+CD45RO+ and CD8+CD45RO+), the source of the pathogenic mediators of psoriasis, while having relatively no effect on naive T-cell populations.

Adolescent↗

PubMed2002

Port of entry--the type III secretion translocon.

Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell. Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon. The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood. In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified. Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions. Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.

Animals↗

PubMed2006

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae.

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.

Amino Acid Sequence↗

PubMed2005

Induction of Nod1 and Nod2 intracellular pattern recognition receptors in murine osteoblasts following bacterial challenge.

Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UV-inactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Selected problems of malaria blood stage immunity.

Both antibody dependent and cell mediated mechanisms contribute to immunity in malaria. The parasites vary in sensitivity to antibody mediated inhibition due to underlying antigenic variation. When Plasmodium falciparum isolates are tested with antibodies from the donor originally harbouring the parasites or with those from other donors, growth inhibition is usually lowest in the autologous combinations. Parasites with decreased sensitivity are also generated in vitro by culturing them for prolonged periods in the presence of certain anti-plasmodial antibodies. When the antibodies are removed, a successive return of sensitivity develops. The decrease in sensitivity to inhibition may either be due to down-regulation of synthesis of the antigen or a selection of parasites with low antigen expression from the heterogeneous original populations. Both T lymphocytes carrying alpha/beta and gamma/delta antigen-receptors play a role in malaria immunity. However, although gamma/delta T cells may expand 40-fold or more in the peripheral immune system in acutely infected humans and also inhibit parasite growth in vitro and in vivo, their relative importance for protection or pathogenicity is presently unclear. Of the two major T cell subsets (CD4+, CD8+) carrying alpha/beta T cell receptors, the role of CD8+ T cells in blood stage infections appears to be limited. Instead, CD4+ T cells are of major importance. These cells comprise at least two functionally different subsets (Th1, Th2), distinguished on the basis of lymphokine secretion. In some rodent malaria models, Th1 cells producing primarily IL2 and IFN gamma give rise to protection in early infection while Th2 cells producing IL4 are essential for parasite clearance in late infection. In other mouse strains, the same parasites induce a strong Th2 response in early infection, resulting in a lethal course. CD4+ T cells of either Th1 or Th2 type also have regulatory functions in human P. falciparum malaria. Most humans living in areas of high endemicity have significantly elevated blood levels of IgE, reflecting a skewing of the underlying T helper cell ratio in favour of Th2, responsible for the switch in immunoglobulin isotypes. Less than 5% of the IgE in malaria represents antibodies to P. falciparum. IgE elevation is highest in patients with severe and particularly cerebral malaria and is frequently associated with an elevation of tumour necrosis factor alpha (TNF). The release of this cytokine from monocytes/macrophages may reflect crosslinking of their low affinity receptors for IgE (CD23) by IgE containing immune complexes from malarial sera. Local overproduction of TNF is considered a major pathogenic mechanism, responsible for fever and tissue lesions in severe malaria. Although TNF overproduction in malaria is generally assumed to be due to direct stimulation of effector cells by certain parasite derived toxins, the present results suggest that IgE elevation constitutes yet another mechanism contributing to the pathogenicity of P. falciparum in human malaria.

Animals↗

PubMed2004

Immunopathogenic mechanisms in psoriasis.

Psoriasis is a common autoimmune skin disease characterized by T cell-mediated hyperproliferation of keratinocytes. The disease has a strong but complex genetic background with a concordance of approximately 60% in monozygotic twins, and recent linkage and high resolution association studies indicate that HLA-Cw*0602 is itself a major susceptibility allele for psoriasis. Patients carrying this allele have been shown to have different clinical features and earlier age of disease onset, and patients homozygous for this allele have about 2.5 times higher disease risk than heterozygotes. Published data indicate that CD8+ T cells may play a major effector role in psoriasis. Epidermal infiltration of predominantly oligoclonal CD8+ T cells, and probably also of CD4+ T cells in the dermis, is a striking feature of chronic psoriasis lesions, indicating that these cells are responding to specific antigens. We argue that CD4+ T cells are essential for initiating and maintaining the pathogenic process of psoriasis but that cross-primed CD8+ T cells are the main effector cells responding to antigens in the HLA-Cw*0602 binding pocket of keratinocytes. It is further proposed that CD8+ T cells are involved in the control of the Th1 polarization, which is observed in psoriasis lesions, through a complex interplay between CD4+, CD8+ T cells and cross-presenting dendritic cells. It is also suggested that spontaneous remissions or fluctuations in disease activity may be determined by a balance within the lesions between effector and suppressor CD4+ and CD8+ T cells.

Autoimmune Diseases↗

PubMed2005

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda.

Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the co-regulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained down regulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immuno-modulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

Animals↗

PubMed2006

Human virus-specific CD8+ T cells: diversity specialists.

Human viruses elicit functionally highly diverse CD8+ T-cell responses. This variation, which manifests in the memory or latency stage, includes differences in expansion requirements, migratory properties, homeostatic maintenance mechanisms, and constitutive effector properties, and it may reflect specific adaptations of the human immune system to challenges posed by individual pathogens. Longitudinal follow-up of individuals after primary virus infections has revealed how divergent virus-specific CD8+ T cells may develop from the initially expanded virus-specific T-cell effector pool. Recent findings have shown that CD8+ T cells reactive toward latent viruses may depend on other mechanisms for their homeostatic maintenance than T cells specific for cleared viruses. Whereas the latter can respond to the homeostatic cytokine interleukin-7 (IL-7), many persistent virus-specific T cells, recognizing latent herpesviruses such as Epstein-Barr virus and cytomegalovirus (CMV), lack IL-7 receptor alpha (IL-7Ralpha) and depend on viral antigens to persist. Finally, CMV is unique in that it generates a vast pool of resting virus-specific T cells with constitutive cytolytic effector function. The putative role of functionally diverse CD8+ T cells in protective immunity to persistent viruses is discussed in this review.

Animals↗

PubMed2006

The type III effectors HsvG and HsvB of gall-forming Pantoea agglomerans determine host specificity and function as transcriptional activators.

Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) induces galls on both beet and gypsophila. The pathogenicity of both pathovars is dependent on the presence of a plasmid harbouring type III secretion system (TTSS) components and effectors. The HsvG TTSS effectors of Pag (HsvG-Pag) and Pab (HsvG-Pab) determine the host specificity of both pathovars on gypsophila. Here we describe a novel HsvG homologue, HsvB, which determines the host specificity of Pag and Pab on beet. HsvG requires two direct amino acid repeats for pathogenicity on gypsophila, whereas one repeat in HsvB is sufficient for pathogenicity on beet. Exchanging repeats between HsvG-Pag and HsvB-Pab resulted in a switch of host specificities. Transient expression of GFP-HsvG or GFP-HsvB fusions in gypsophila, beet or melon leaves showed that HsvG and HsvB were localized to the nuclei of host and non-host plants. A yeast one-hybrid assay revealed that a single repeat of HsvG or HsvB was sufficient to activate transcription. By employing random binding-site selection and gel-shift assay HsvG was demonstrated to be a double-stranded DNA-binding protein with an ACACC/aAA consensus binding site. These results suggest that HsvG and HsvB are host-specificity determinants and bear the potential to affect the host transcriptional machinery.

Bacterial Proteins↗

PubMed2004

Mechanisms for impaired effector function in alveolar macrophages from marijuana and cocaine smokers.

Lung macrophages provide a first line of host defense against inhaled pathogens and their function is impaired in the lungs of inhaled substance abusers. In order to investigate the mechanism for this impairment, alveolar macrophages (AM) were recovered from nonsmokers (NS), regular tobacco smokers (TS), marijuana smokers (MS), or crack cocaine smokers (CS), and evaluated for their production of nitric oxide (NO) and the role of NO as an antimicrobial effector molecule. AM from NS and TS efficiently killed Staphylococcus aureus and their antibacterial activity correlated closely with the production of nitrite and the expression of mRNA encoding for inducible nitric oxide synthase (iNOS). In contrast, AM collected from MS and CS exhibited limited antimicrobial activity that was not affected by an inhibitor of iNOS, or associated with expression of iNOS. Treatment with either granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-gamma restored the ability of these cells to produce NO and to kill bacteria. These findings confirm a significant role for NO as an antibacterial effector molecule used by normal human AM and suggest that this host defense mechanism is suppressed by habitual exposure to inhaled marijuana or crack cocaine in vivo.

Adult↗

PubMed2002

c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.

The human pathogen Helicobacter pylori colonizes the mucous layer of the stomach. During parasitic infection, freely swimming bacteria adhere to the gastric epithelial cells and trigger intracellular signalling pathways. This process requires the translocation of the effector protein CagA into the host cell through a specialized type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine residues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C-terminal half of the protein and contains the five-amino-acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c-Src and Lyn, account for most of the CagA-specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor-like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.

Amino Acid Motifs↗

PubMed2001

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. pyogenes NAD-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.

Adenosine Diphosphate Ribose↗

PubMed1996

Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Amino Acid Sequence↗