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PubMed2003

The Hrp pilus: learning from flagella.

Plant pathogenic bacteria deliver avirulence and virulence effector proteins into plant cells via the hrp-gene-encoded type III secretion system. A key component of this secretion system is a surface appendage called the Hrp pilus. Recent results suggest that the Hrp pilus serves as a conduit for type III protein secretion and that it is assembled in a manner similar to the flagellum. The Hrp pilus is likely to be the functional equivalent of the needle extension, assembled by type III secretion systems of mammalian pathogenic bacteria.

Animals↗

PubMed1998

Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis.

CD4+ memory T cells coordinate immune responses against viruses and other pathogens via the Ag-induced secretion of potent effector cytokines. The efficacy of these responses depends on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. Here, we provide evidence that heterogeneity in Ag triggering thresholds constitutes an additional critical determinant of memory T cell function. Using a novel assay that allows single-cell detection of Ag-specific T cell cytokine production, we demonstrate that CMV-specific CD4+ memory cells from human peripheral blood display pronounced differences in their costimulatory requirements for Ag-induced triggering of IFN-gamma and IL-2 secretion, ranging from cells that trigger with little costimulation (e.g., resting APC alone) to cells requiring potent costimulation through multiple pathways (resting APC plus multiple costimulatory mAbs, or activated APC). These differences in costimulatory requirements are independent of clonal differences in TCR signaling intensity, consistent with an intrinsic activation-threshold heterogeneity that is "downstream" from the TCR. Thus, "effective" frequencies of Ag-specific CD4+ memory T cells appear to depend on the activation status of available APC, a dependence that would allow the immune system to rapidly adjust the number of functional Ag-specific memory T cells in a particular effector site according to local conditions.

Antibodies, Monoclonal↗

PubMed2006

Potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic Escherichia coli-injected effector proteins.

Enteropathogenic Escherichia coli (EPEC) induces a severe watery diarrhea responsible for several hundred thousand infant deaths each year by a process correlated with the loss (effacement) of absorptive microvilli. Effacement is linked to the locus of enterocyte effacement pathogenicity island that encodes an "injection system," "effector" proteins, and the Intimin outer membrane protein. Here, we reveal that effacement (i) is a two-step process, (ii) requires the cooperative action of three injected effectors (Map, EspF, and Tir) as well as Intimin, and (iii) leads to the retention, not release (into the extracellular milieu), of the detached microvillar material. We also discover that EPEC rapidly inactivates the sodium-d-glucose cotransporter (SGLT-1) by multiple mechanisms. Indeed, the finding that one mechanism occurs more rapidly than microvilli effacement provides a plausible explanation for the rapid onset of severe watery diarrhea, given the crucial role of SGLT-1 in the daily uptake of approximately 6 liters of fluids from the normal intestine. The importance of SGLT-1 in the disease process is supported by severe EPEC diarrheal cases being refractory to oral rehydration therapy (dependent on SGLT-1 function). Moreover, the identification of effector activities that alter microvilli structure and SGLT-1 function provides new tools for studying the underlying regulatory processes.

Adhesins, Bacterial↗

PubMed2004

The anaphylatoxins bridge innate and adaptive immune responses in allergic asthma.

The complement system has long been recognized for its role as a lytic effector system that protects against microbial pathogens, as well as for its role in mediating acute and chronic inflammatory responses. Many of the inflammatory sequelae of complement activation can be related to the complement cleavage fragments C3a and C5a, the so-called anaphylatoxins (ATs). Cloning and subsequent gene targeting of their corresponding receptors, as well as generation of specific C3a and C5a inhibitors, have fueled new interest in studies aimed at defining the roles of the anaphylatoxins in inflammatory diseases. Traditionally, the anaphylatoxins have been considered mediators of end-stage effector mechanisms. However, recent data from animal models of allergic asthma suggest that C3a and C5a provide a critical link between innate and adaptive immunity. This review is aimed at outlining our current knowledge of when and where anaphylatoxins contribute to and control the development of allergic asthma. The accumulated data suggest a model in which C3a and C5a play important but opposing roles during allergen-induced T-cell polarization: C3a promotes Th2 responses, whereas C5a prevents Th2 polarization. During the effector phase, both anaphylatoxins trigger the inflammatory response and contribute to bronchoconstriction.

Allergens↗

PubMed2002

Immune surveillance and effector functions of CCR10(+) skin homing T cells.

Skin homing T cells carry memory for cutaneous Ags and play an important sentinel and effector role in host defense against pathogens that enter via the skin. CCR10 is a chemokine receptor that is preferentially expressed among blood leukocytes by a subset of memory CD4 and CD8 T cells that coexpress the skin-homing receptor cutaneous lymphocyte Ag (CLA), but not the gut-homing receptor alpha(4)beta(7). Homing and chemokine receptor coexpression studies detailed in this study suggest that the CLA(+)/CCR10(+) memory CD4 T cell population contains members that have access to both secondary lymphoid organ and skin compartments; and therefore, can act as both "central" and "effector" memory T cells. Consistent with this effector phenotype, CLA(+)/CCR10(+) memory CD4 T cells from normal donors secrete TNF and IFN-gamma but minimal IL-4 and IL-10 following in vitro stimulation. Interactions of CCR10 and its skin-associated ligand CC ligand 27 may play an important role in facilitating memory T cell entry into cutaneous sites during times of inflammation.

Animals↗

PubMed2002

Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37.

Effectors of the innate immune system, the anti-bacterial peptides, have pivotal roles in preventing infection at epithelial surfaces. Here we show that proteinases of the significant human pathogens Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and Streptococcus pyogenes, degrade the antibacterial peptide LL-37. Analysis by mass spectrometry of fragments generated by P. aeruginosa elastase in vitro revealed that the initial cleavages occurred at Asn-Leu and Asp-Phe, followed by two breaks at Arg-Ile, thus inactivating the peptide. Proteinases of the other pathogens also degraded LL-37 as determined by SDS-PAGE. Ex vivo, P. aeruginosa elastase induced LL-37 degradation in human wound fluid, leading to enhanced bacterial survival. The degradation was blocked by the metalloproteinase inhibitors GM6001 and 1, 10-phenantroline (both of which inhibited P. aeruginosa elastase, P. mirabilis proteinase, and E. faecalis gelatinase), or the inhibitor E64 (which inhibited S. pyogenes cysteine proteinase). Additional experiments demonstrated that dermatan sulphate and disaccharides of the structure [DeltaUA(2S)-GalNAc(4,6S)], or sucroseoctasulphate, inhibited the degradation of LL-37. The results indicate that proteolytic degradation of LL-37 is a common virulence mechanism and that molecules which block this degradation could have therapeutic potential.

Amino Acid Sequence↗

PubMed2004

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge.

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Acid Anhydride Hydrolases↗

PubMed2006

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A.

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Adenylyl Cyclases↗

PubMed2002

Functional plasticity of an antigen-specific memory CD4 T cell population.

The protective nature of memory immune responses is attributed largely to terminally differentiated memory T cells that retain memory of the antigen via the antigen receptor and memory of the effector functions that initially cleared the pathogen. It is not known whether a given population of antigen-specific memory T cells is endowed with functional flexibility to provide protective responses against antigens reencountered in different immunological contexts. Here, we examine functional properties of influenza hemagglutinin (HA)-specific memory CD4 T cells recovered from adoptive hosts that received in vitro-activated HA-specific T cell receptor-transgenic CD4 T cells 2 months to 1 year previously. We demonstrate that this HA-specific memory CD4 T cell population bearing a clonal T cell receptor can produce predominantly T helper 1 or T helper 2 effector cytokines depending on the nature of the recall stimulus. Our findings reveal remarkable functional plasticity within an antigen-specific memory T cell population and have direct implications for modulating memory T cell function in vaccine design and treatments for autoimmune diseases.

Adoptive Transfer↗

PubMed1994

Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide.

Like many pathogens that undergo an intravascular stage of development, larvae of the helminth parasite Schistosoma mansoni migrate through the blood vessels, where they are in close contact with endothelial cells. In vitro exposure of murine endothelial cells to various cytokines (interferon gamma, tumor necrosis factor alpha, and interleukin 1 alpha or 1 beta) resulted in their activation to kill schistosomula through an arginine-dependent mechanism involving production of nitric oxide (NO). Cytokine-treated endothelial cells showed increased expression of mRNA for the inducible form of the NO synthase, and both NO production and larval killing were suppressed by treatment with competitive inhibitors. The effector function of cytokine-treated endothelial cells was similar to that of activated inflammatory tissue macrophages, although activation appeared to be differentially regulated in these two cell types. Activated endothelial cells killed older (18-day) forms of the parasite, such as those currently thought to be a primary target of immune elimination in the lungs of mice previously vaccinated with radiation-attenuated cercariae, as well as newly transformed larvae. In C57BL/6 mice, which become resistant to S. mansoni infection as a result of vaccination with irradiated cercariae, endothelial cell morphology characteristic of activation was observed in the lung by 1-2 weeks after challenge infection. Similar endothelial cell changes were absent in P-strain mice, which do not become resistant as a result of vaccination. Together, these observations indicate that endothelial cells, not traditionally considered to be part of the immune system, may play an important role in immunity to S. mansoni and, by means of NO-dependent killing, could serve as effectors of resistance to other intravascular pathogens.

Amino Acid Oxidoreductases↗

PubMed2006

[Type IV secretion system and their effectors: an update].

Subversion of eukaryotic hosts by bacterial pathogens requires specialized macromolecules secretion systems delivering virulence factors either into the environment or directly into host cells. Transport of molecules across bacterial and eukaryotic membranes is a process requiring multi-component machineries called secretion systems. This review focuses on the Type IV secretion system. This complex is required for genetic exchange (DNA transport) and secretion of effectors (proteins, macromolecules, DNA-proteins complex) into target cells. They transport a wide variety of substrates including large DNA/protein complexes, multi protein toxins, or individual proteins. We describe recent advances on the structure and the function of this secretion system, their effectors and their effects on the functions of eukaryotic cell.

Bacteria↗

PubMed2001

Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis.

Multiple periodontal pathogens sequentially colonize the subgingival niche during the conversion from gingivitis to destructive periodontal disease. An animal model of sequential immunization with key periodontal pathogens has been developed to determine whether T- and B-lymphocyte effector functions are skewed and fail to protect the host from pathogenic challenge. The present study was performed to evaluate the immunomodulatory effect of exposure to Fusobacterium nucleatum prior to Porphyromonas gingivalis. Group 1 (control) mice were immunized with phosphate-buffered saline, group 2 were immunized with F. nucleatum prior to P. gingivalis and group 3 were immunized with P. gingivalis alone. All the T-cell clones derived from group 2 demonstrated type 2 helper T-cell clone (Th2 subsets), whereas those from group 3 mice demonstrated Th1 subsets. Exposure of mice to F. nucleatum prior to P. gingivalis interfered with the opsonophagocytosis function of sera against P. gingivalis. In adoptive T-cell transfer experiments, in vivo protective capacity of type 2 helper T-cell clones (Th2) from group 2 was significantly lower than type 1 helper T-cell clones (Th1) from group 3 against the lethal dose infection of P. gingivalis. Western blot analysis indicated a different pattern of recognition of P. gingivalis fimbrial proteins between sera from group 2 and group 3. In conclusion, these studies suggest that exposure of a host to F. nucleatum prior to the periodontal pathogen P. gingivalis modulates the host immune responses to P. gingivalis at the humoral, cellular and molecular levels.

Adjuvants, Immunologic↗

PubMed2004

Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter.

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis: The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.

Adenylyl Cyclases↗

PubMed2005

CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is an intestinal attaching and effacing pathogen that utilizes a type III secretion system (T3SS) for the delivery of effectors into host cells. The chaperone CesT has been shown to bind and stabilize the type III translocated effectors Tir and Map in the bacterial cytoplasm prior to their delivery into host cells. In this study we demonstrate a role for CesT in effector recruitment to the membrane embedded T3SS. CesT-mediated effector recruitment was dependent on the presence of the T3SS membrane-associated ATPase EscN. EPEC DeltacesT carrying a C-terminal CesT variant, CesT(E142G), exhibited normal cytoplasmic Tir stability function, but was less efficient in secreting Tir, further implicating CesT in type III secretion. In vivo co-immunoprecipitation studies using CesT-FLAG containing EPEC lysates demonstrated that CesT interacts with Tir and EscN, consistent with the notion of CesT recruiting Tir to the T3SS. CesT was also shown to be required for the efficient secretion of several type III effectors encoded within and outside the locus of enterocyte effacement (LEE) in addition to Tir and Map. Furthermore, a CesT affinity column was shown to specifically retain multiple effector proteins from EPEC culture supernatants. These findings indicate that CesT is centrally involved in recruiting multiple type III effectors to the T3SS via EscN for efficient secretion, and functionally redefine the role of CesT in multiple type III effector interactions.

Adenosine Triphosphatases↗

PubMed2004

Proteases in pathogenesis and plant defence.

Plant pathogens deliver a variety of virulence factors to host cells to suppress basal defence responses and create suitable environments for their propagation. Plants have in turn evolved disease resistance genes whose products detect the virulence factors as a signal of invasion and activate effective defence responses. Understanding how a virulence effector contributes to virulence on susceptible hosts but becomes an avirulence factor that triggers defence responses on resistance hosts has been a major focus in plant research. Recent studies have shown that a growing list of pathogen-encoded effectors functions as proteases that are secreted into plant cells to modify host proteins. In addition, several plant proteases have been found to function in activation of the defence mechanism. These findings reveal that post-translational modification of host proteins through proteolytic processing is a widely used mechanism in regulating the plant defence response.

Bacterial Proteins↗

PubMed1999

Inducible anti-parasitic effector mechanisms in human uroepithelial cells: tryptophan degradation vs. NO production.

In murine cells the most important effector mechanism directed against the intracellular pathogen Toxoplasma gondii is the production of toxic nitrogen oxides. In contrast the induction of the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO) has been described to be the most effective anti-parasitic mechanism in most human cells. In this report we analysed IDO induction and NO production in the human uroepithelial carcinoma cell line RT4. We found that after stimulation with IFN-gamma these cells were able to restrict toxoplasma growth. This was due to an activation of IDO, and the anti-parasitic effect mediated by RT4 cells was abrogated by the addition of L-tryptophan. In addition we found that the costimulation of RT4 cells with IL-1 and IFN-gamma results in the production of nitric oxide, and that in RT4 cells stimulated with both these cytokines, IDO activity and toxoplasmostasis was lower than in cells stimulated with IFN-gamma alone. This IL-1-mediated inhibition of IFN-gamma-induced IDO activity and toxoplasmostasis could be blocked by monomethyl L-arginine, an inhibitor of NO production. We therefore conclude that the induction of indolamine 2,3-dioxygenase activity in human cells is a very important effector mechanism directed against Toxoplasma gondii, and that in human cells the production of NO might be involved in the regulation of IDO activity.

Animals↗

PubMed2002

Genetically modified Streptococcus mutans for the prevention of dental caries.

There are many examples of positive and negative interactions between different species of bacteria inhabiting the same ecosystem. This observation provides the basis for a novel approach to preventing microbial diseases called replacement therapy. In this approach, a harmless effector strain is permanently implanted in the host's microflora. Once established, the presence of the effector strain prevents the colonization or outgrowth of a particular pathogen. In the case of dental caries, replacement therapy has involved construction of an effector strain called BCS3-L1, which was derived from a clinical Streptococcus mutans isolate. Recombinant DNA technology was used to delete the gene encoding lactate dehydrogenase in BCS3-L1 making it entirely deficient in lactic acid production. This effector strain was also designed to produce elevated amounts of a novel peptide antibiotic called mutacin 1140 that gives it a strong selective advantage over most other strains of S. mutans. In laboratory and rodent model studies, BCS3-L1 was found to be genetically stable and to produce no apparent deleterious side effects during prolonged colonization. BCS3-L1 was significantly less cariogenic than wild-type S. mutans in gnotobiotic rats, and it did not contribute at all to the cariogenic potential of the indigenous flora of conventional Sprague-Dawley rats. And, its strong colonization properties indicated that a single application of the BCS3-L1 effector strain to human subjects should result in its permanent implantation and displacement over time of indigenous, disease-causing S. mutans strains. Thus, BCS3-L1 replacement therapy for the prevention of dental caries is an example of biofilm engineering that offers the potential for a highly efficient, cost effective augmentation of conventional prevention strategies. It is hoped that the eventual success of replacement therapy for the prevention of dental caries will stimulate the use of this approach in the prevention of other bacterial diseases.

Dental Caries↗

PubMed2002

Envelope instability in DNA adenine methylase mutants of Salmonella enterica.

Mutants of Salmonella enterica serovar Typhimurium lacking DNA adenine (Dam) methylase show reduced secretion of invasion effectors encoded in the Salmonella-pathogenicity island 1 (SPI-1). Concomitant with this alteration, a high number and quantity of extracellular proteins are detected in cultures of Dam(-) mutants. This study shows by subcellular fractionation analysis that the presence of numerous extracellular proteins in cultures of Dam(-) mutants is linked to an exacerbated release of membrane particulate material. The membrane 'leaky' phenotype and the impaired functionality of type III secretion systems were, however, unrelated since exacerbated release of proteins to the medium was evident in Dam(-) strains carrying mutations in either SPI-1 (invA, invJ) or flagellar (flhD) genes. This result supports the view that Dam methylation controls a plethora of cellular processes. Electron microscopy analysis demonstrated that the accumulation of membrane particulate material occurs preferentially as vesicles in stationary cultures of Dam(-) strains. In addition, a reduction in the relative amount of peptidoglycan-associated lipoprotein (PAL), TolB, OmpA and murein lipoprotein (Lpp) bound to peptidoglycan was observed in actively growing Dam(-) mutants. The existence of an envelope defect was further confirmed by the increased sensitivity to deoxycholate exhibited by Dam(-) mutants, mostly during exponential growth. Unexpectedly, lack of Dam methylation neither increased envelope instability nor impaired the association of PAL-Tol-Lpp proteins to the peptidoglycan in Escherichia coli. Accordingly, E. coli Dam(-) mutants did not show sensitivity to deoxycholate. Altogether, these results indicate that, besides its role in modulating the secretion of effectors by the SPI-1-encoded type III apparatus, Dam methylation controls cell envelope integrity in S. enterica.

Bacterial Proteins↗