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PubMed2004

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins.

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

Bacterial Outer Membrane Proteins↗

PubMed1998

Divergent roles for Fc receptors and complement in vivo.

Recent results obtained in mice deficient in either FcRs or complement have revealed distinct functions for these two classes of molecules. While each is capable of interacting with antibodies or immune complexes, the two systems mediate distinct biological effector responses. Complement-deficient mice are unable to mediate innate immune responses to several bacterial pathogens and bacterial toxins, yet respond normally to the presence of cytotoxic antibodies and pathogenic immune complexes. In contrast, FcR-deficient mice display no defects in innate immunity or susceptibility to a variety of pathogens, yet they are unable to mediate inflammatory responses to cytotoxic IgG antibodies or IgG immune complexes, despite the presence of a normal complement system. These results lead to the surprising conclusion that these two systems have evolved distinct functions in host immunity, with complement and its receptors mediating the interaction of natural antibodies (IgM) with pathogens to effect protection, while FcRs couple the interaction of IgG antibodies to effector cells to trigger inflammatory sequelae. These results necessitate a fundamental revision of the role of these antibody-binding systems in the immune response.

Animals↗

PubMed1999

Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

Animals↗

PubMed2006

Delayed expansion and contraction of CD8+ T cell response during infection with virulent Salmonella typhimurium.

Ag presentation to CD8(+) T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (approximately 7 days), resistant mice (129 x 1SvJ) harbor a chronic infection lasting approximately 60-90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8(+) T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62L(high)IL-7Ralpha(high)CD44(high)) CD8(+) T cells. However, by day 14-21, majority of the primed CD8(+) T cells display an effector phenotype (CD62L(low)IL-7Ralpha(low)CD44(high)). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62L(low)IL-7Ralpha(high)CD44(high)) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8(+) T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8(+) T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8(+) T cell recognition, conferring a survival advantage to the pathogen.

Animals↗

PubMed2006

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen.

We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8(+) effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8(+) effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens.

Animals↗

PubMed2004

Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism.

Infection by the Gram-negative bacterial pathogen Shigella flexneri depends on its ability to invade host cells. Bacterial engulfment requires a functional type III secretion system (TTSS) allowing the translocation into host cells of bacterial effectors that activate cell-signaling cascades. We demonstrated previously that specialized lipid membrane domains enriched in cholesterol and sphingolipids (rafts) are involved during early steps of invasion, namely in binding and host cell entry. In this study, we addressed the issue of contact-mediated secretion by the TTSS. We show that contact-mediated and TTSS-induced hemolysis depend on the presence of cholesterol on the host cell surface. We found that purified detergent resistant membranes were able to activate TTSS. Finally, we found that artificial liposomes, devoid of proteins, were able to activate the TTSS but only when their composition mimicked that of lipid rafts. Altogether, these data indicate that specific lipid packing can trigger contact-mediated secretion by S. flexneri.

Animals↗

PubMed2006

The enteropathogenic Escherichia coli EspF effector molecule inhibits PI-3 kinase-mediated uptake independently of mitochondrial targeting.

Delivery of effector molecules into LMme(v) macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinase-dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE- and non-LEE-encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE-encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI-3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245-1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS-defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C-terminal domain possesses a polyproline motif associated with protein-protein interactions. We demonstrate that EspF-mediated inhibition of PI-3 kinase-dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N-terminal domain with and (iii) the C-terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI-3 kinase-dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI-3 kinase-dependent uptake process.

Adhesins, Bacterial↗

PubMed2005

Regulatory roles for cytokine-producing B cells in infection and autoimmune disease.

Recent experiments have revealed that B cells can regulate the course of immune responses to pathogens and autoantigens by antibody-independent mechanisms. One antibody-independent function of B cells is to produce cytokines. In this review we describe the identification of IL-10-producing 'regulatory' B cells as well as IFNgamma-producing 'effector' Bel cells and IL-4-producing 'effector' Be2 cells. We discuss the roles of antigen, pathogen-derived molecules and T cell and dendritic cell-derived factors in regulating the differentiation of mature B cells into cytokine-producing effector B cells. We also review the recent experiments showing that B cell-derived cytokines play pathologic as well as protective roles in immune responses to autoantigens, and demonstrate that cytokine-producing B cells play unexpectedly complex and potentially opposing roles in autoimmune disease.

Animals↗

PubMed2001

Mechanisms of T cell receptor antagonism: implications in the treatment of disease.

The adaptive immune response is often required for the successful clearing of infectious pathogens. Antigen presenting cells (APC) present peptide antigens derived from pathogens to T cells via major histocompatibility complex (MHC) molecules. T cells then become activated and differentiate into effector cells with the capacity to kill infected cells or to induce an anti-pathogen antibody response. In autoimmunity, this T cell response is directed against self-antigens and often leads to deleterious effects on specific tissues. Likewise, T cell responses to allogeneic MHC molecules in transplants also leads to pathology. By introducing subtle changes in the antigenic peptide amino acid content, T cell activation can be inhibited, thereby preventing T cell effector functions. This strategy of TCR antagonism has been used successfully in vitro and in vivo to inhibit models of autoimmunity and allorecognition. In addition, a variety of pathogens that often result in chronic disease following infection, also have seemingly evolved natural mechanisms to inhibit T cell responses by antagonism. These microorganisms express natural variants of certain proteins, that when presented to T cells have the capacity to specifically inhibit T cell responses by functioning as antagonists or by modulating the nature of the T cell response. The understanding of how pathogens mediate this inhibition in vivo will be beneficial to ongoing studies in both autoimmunity and transplantation aimed at suppressing the harmful immune response, thereby controlling disease. TCR antagonism seems to have the potential to be used therapeutically to prevent or inhibit an undesired T cell response that will ultimately lead to disease.

Animals↗

PubMed2005

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.

Animals↗

PubMed2005

Effector mechanisms of nonsuppurative destructive cholangitis in graft-versus-host disease and allograft rejection.

The biliary epithelium provides a physical barrier to ascending infection from the gastrointestinal tract and is also involved in actively regulating the immune response to invading pathogens. Cholangiocytes secrete chemokines and express adhesion molecules that attract effector leukocytes and promote the clearance of infected cells. However in the context of transplantation these properties make cholangiocytes targets for allogeneic cytotoxic T cells, and both graft-versus-host disease and liver allograft rejection are characterized by destruction of intrahepatic bile ducts by infiltrating lymphocytes. The mechanisms of cholangiocyte killing are complex but involve activation of apoptosis by the granzyme/perforin pathway and by activation of death receptors belonging to the tumor necrosis factor (TNF) receptor superfamily, most notably Fas. Fas-dependent apoptosis is carefully regulated by cooperative interactions with other TNF receptors, particularly CD40, that act to amplify autocrine and paracrine expression of Fas ligand and Fas-mediated killing. A better understanding of the molecular control of these processes may explain why bile duct loss continues despite conventional immunosuppression in the vanishing bile duct syndromes, and lead to novel therapies aimed at switching off the chronic inflammatory response and protecting cholangiocytes from apoptosis.

Apoptosis↗

PubMed2006

The pathogen-host interactions database (PHI-base) provides insights into generic and novel themes of pathogenicity.

Fungal and oomycete pathogens of plants and animals are a major global problem. In the last 15 years, many genes required for pathogenesis have been determined for over 50 different species. Other studies have characterized effector genes (previously termed avirulence genes) required to activate host responses. By studying these types of pathogen genes, novel targets for control can be revealed. In this report, we describe the Pathogen-Host Interactions database (PHI-base), which systematically compiles such pathogenicity genes involved in pathogen-host interactions. Here, we focus on the biology that underlies this computational resource: the nature of pathogen-host interactions, the experimental methods that exist for the characterization of such pathogen-host interactions as well as the available computational resources. Based on the data, we review and analyze the specific functions of pathogenicity genes, the host-specific nature of pathogenicity and virulence genes, and the generic mechanisms of effectors that trigger plant responses. We further discuss the utilization of PHI-base for the computational identification of pathogenicity genes through comparative genomics. In this context, the importance of standardizing pathogenicity assays as well as integrating databases to aid comparative genomics is discussed.

Computational Biology↗

PubMed2006

Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum.

In many plant and animal bacterial pathogens, the Type III secretion system (TTSS) that directly translocates effector proteins into the eukaryotic host cells is essential for the development of disease. In all species studied, the transcription of the TTSS and most of its effector substrates is tightly regulated by a succession of consecutively activated regulators. However, the whole genetic programme driven by these regulatory cascades is still unknown, especially in bacterial plant pathogens. Here, we have characterised the programme triggered by HrpG, a host-responsive regulator of the TTSS activation cascade in the plant pathogen Ralstonia solanacearum. We show through genome-wide expression analysis that, in addition to the TTSS, HrpG controls the expression of a previously undescribed TTSS-independent pathway that includes a number of other virulence determinants and genes likely involved in adaptation to life in the host. Functional studies revealed that this second pathway co-ordinates the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. We provide experimental evidence that these activities contribute to pathogenicity. We also show that the ethylene produced by R. solanacearum is able to modulate the expression of host genes and can therefore interfere with the signalling of plant defence responses. These results provide a new, integrated view of plant bacterial pathogenicity, where a common regulator activates synchronously upon infection the TTSS, other virulence determinants and a number of adaptive functions, which act co-operatively to cause disease.

Adaptation, Physiological↗

PubMed2003

The versatile bacterial type IV secretion systems.

Bacteria use type IV secretion systems for two fundamental objectives related to pathogenesis--genetic exchange and the delivery of effector molecules to eukaryotic target cells. Whereas gene acquisition is an important adaptive mechanism that enables pathogens to cope with a changing environment during invasion of the host, interactions between effector and host molecules can suppress defence mechanisms, facilitate intracellular growth and even induce the synthesis of nutrients that are beneficial to bacterial colonization. Rapid progress has been made towards defining the structures and functions of type IV secretion machines, identifying the effector molecules, and elucidating the mechanisms by which the translocated effectors subvert eukaryotic cellular processes during infection.

Bacterial Proteins↗

PubMed2005

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens.

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.

Antigens, Bacterial↗

PubMed2005

The development of the mucosal immune system pre- and post-weaning: balancing regulatory and effector function.

The mucosal immune system fulfils the primary function of defence against potential pathogens that may enter across vulnerable surface epithelia. However, a secondary function of the intestinal immune system is to discriminate between pathogen-associated and 'harmless' antigens, expressing active responses against the former and tolerance to the latter. Control of immune responses appears to be an active process, involving local generation of IgA and of regulatory and/or regulated T lymphocytes. Two important periods of maximum exposure to novel antigens occur in the young animal, immediately after birth and at weaning. In both cases the antigenic composition of the intestinal contents can shift suddenly, as a result of a novel diet and of colonisation by novel strains and species of bacteria. Changes in lifestyles of man, and husbandry of animals, have resulted in weaning becoming much more abrupt than previously in evolution, increasing the number of antigens that must be simultaneously evaluated by neonates. Thus, birth and weaning are likely to represent hazard and critical control points in the development of appropriate responses to pathogens and harmless dietary and commensal antigens. Neonates are born with relatively undeveloped mucosal immune systems. At birth this factor may prevent both expression of active immune responses and development of tolerance. However, colonisation by intestinal flora expands the mucosal immune system in antigen-specific and non-specific ways. At weaning antibody to fed proteins can be detected, indicating active immune responses to fed proteins. It is proposed that under normal conditions the ability of the mucosal immune system to mount active responses to foreign antigens develops simultaneously with the ability to control and regulate such responses. Problems arise when one or other arm of the immune system develops inappropriately, resulting in inappropriate effector responses to harmless food proteins (allergy) or inadequate responses to pathogens (disease susceptibility).

Animals↗

PubMed2004

An unexpected role for hypoxic response: oxygenation and inflammation.

The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant cells in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.

Animals↗