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PubMed2003

Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors.

Polymorphonuclear leukocytes (PMNs) are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites and killing pathogenic microbes. We addressed the role of chemokine receptor desensitization induced by G-protein-coupled receptor kinases (GRKs) in the feedback control of PMN migration. We show that the chemokine macrophage inflammatory protein-2 (MIP-2) induces GRK2 and GRK5 expression in PMNs through phosphoinositide-3-kinase (PI3K)-gamma signaling. We also show that lipopolysaccharide (LPS)-activated signaling through the Toll-like receptor (TLR)-4 pathway transcriptionally downregulates the expression of GRK2 and GRK5 in response to MIP-2. The reduced expression of GRKs lowers chemokine receptor desensitization and markedly augments the PMN migratory response. These data indicate that TLR4 modulation of PMN surface chemokine receptor expression subsequent to the downregulation of GRK2 and GRK5 expression is a critical determinant of PMN migration.

Animals↗

PubMed1999

Mutational analysis of the Pseudomonas syringae pv. tomato hrpA gene encoding Hrp pilus subunit.

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus-dependent autoagglutination of the bacteria. The majority of insertions in the amino-terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy-terminus appeared to be needed for pilus assembly. Insertions in the 5' non-translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

5' Untranslated Regions↗

PubMed2000

HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant.

The interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III-secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using non-polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non-conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp-dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.

Bacterial Proteins↗

PubMed2002

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

Amino Acid Sequence↗

PubMed1999

Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system.

"Natural" Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.

Animals↗

PubMed2001

Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro.

Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN's or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

Antigens, CD↗

PubMed2000

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma and/or lipopolysaccharide in murine macrophage cell lines. Role of tumor necrosis factor-alpha.

Macrophages respond to infection or injury by changing from a "resting" cellular phenotype to an "activated" state defined by the expression of various cytotoxic effector functions. Regulation of the transition from a resting to an activated state is effected by cytokine and/or pathogenic signals. Some signals do not directly induce activation, but instead "prime" the macrophage to respond more vigorously to a second signal. One example of this priming phenomenon involves induction of nitric oxide (NO) synthesis by the enzyme nitric oxide synthase (NOS2). Our experiments indicate that low doses (1-5 Gy) of ionizing radiation can enhance the induction of enzymatically active NOS2 by IFN-gamma or LPS in J774.1 and RAW264.7 murine macrophage cell lines. Radiation alone did not produce this induction, rather, it was effective as a priming signal; cells exposed to radiation produced more NO when a second signal, either IFN-gamma or LPS, was applied 24 h later.

Animals↗

PubMed2002

Immunity-related genes and gene families in Anopheles gambiae.

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

Alternative Splicing↗

PubMed1997

Distinct mechanisms of immunosuppression as a consequence of major surgery.

Altered host defense mechanisms after major surgery or trauma are considered important for the development of infectious complications and sepsis. In the present study, we demonstrate that major surgery results in a severe defect of T-lymphocyte proliferation and cytokine secretion in response to coligation of the antigen receptor complex and CD28. During the early postoperative course, reduced cytokine secretion was observed for interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha, which are associated with the Th1 phenotype of helper T lymphocytes, and for IL-4, the index cytokine of Th2 cells. During the late postoperative course, T-cell cytokine secretion increased to normal levels. Production of the anti-inflammatory cytokine IL-10 was altered, with different kinetics being selectively elevated during the late postoperative course. In contrast, the capacity of peripheral blood monocytes to present bacterial superantigens and to stimulate T-cell proliferation was normal or enhanced after surgery despite a significant loss of cell surface HLA-DR molecules. Thus, the level of major histocompatibility complex class II protein expression does not appear to predict the antigen-presenting capacity of monocytes obtained from surgical patients with uneventful postoperative recovery. Secretion of IL-1beta and IL-10 by endotoxin-stimulated peripheral blood monocytes was increased at different time points after surgery. Major surgery therefore results in a distinct pattern of immune defects with a predominant defect in the T-cell response to T-cell receptor- and CD28 coreceptor-mediated signals rather than an impaired monocyte antigen-presenting capacity. Suppression of T-cell effector functions during the early phase of the postoperative course may define a state of impaired defense against pathogens and increased susceptibility to infection and septic complications.

Aged↗

PubMed1999

YopJ of Yersinia spp. is sufficient to cause downregulation of multiple mitogen-activated protein kinases in eukaryotic cells.

Pathogenic Yersinia spp. utilize a plasmid-encoded type III secretion system to deliver a set of Yop effector proteins into eukaryotic cells. Previous studies have shown that the effector YopJ is required for Yersinia to cause downregulation of the mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) 1 and 2 in infected macrophages. Here we demonstrate that YopJ is sufficient to cause downregulation of multiple MAP kinases in eukaryotic cells. Cellular fractionation experiments confirmed that YopJ is delivered into the cytoplasmic fraction of macrophages by the type III system. Production of YopJ in COS-1 cells by transfection significantly reduced (5- to 10-fold) activation of JNK, p38, and ERK in response to several different stimuli, including serum and tumor necrosis factor alpha. JNK activation mediated by RacV12, an activated mutant of Rac1, was also blocked by YopJ in COS-1 cells, indicating that YopJ acts downstream of this small GTPase to downregulate MAP kinase signaling. Analysis of transfected COS-1 cells by immunofluorescence microscopy revealed that YopJ is recruited from the cytoplasmic compartment to the cell periphery in response to stimuli (e.g., serum) that induce membrane ruffling. These data indicate that YopJ functions as a "MAP kinase toxin" to selectively block nuclear responses that are triggered by Yersinia-host cell interaction.

Animals↗

PubMed2000

A regulatory role for interleukin 4 in differential inflammatory responses in the lung following infection of mice primed with Th1- or Th2-inducing pertussis vaccines.

Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge with Bordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course of B. pertussis infection of naïve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1beta (IL-1beta) in the lungs. In contrast, neutrophil infiltration and IL-1beta production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs following B. pertussis infection of IL-4-defective (IL-4(-/-)) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1beta, IL-6, and IL-1ra in Pa-immunized IL-4(-/-) mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge with B. pertussis and implicate IL-4 as an important regulator of inflammatory-cell recruitment.

Animals↗

PubMed2002

The Salmonella enterica serotype typhimurium effector proteins SipA, SopA, SopB, SopD, and SopE2 act in concert to induce diarrhea in calves.

Salmonella enterica serotype Typhimurium requires a functional type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves. Salmonella serotype Typhimurium strains having mutations in sptP, avrA, sspH1, or slrP induced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wild type. In contrast, mutations in sipA, sopA, sopB, sopD, or sopE2 significantly reduced fluid accumulation in bovine ligated ileal loops at 8 h postinfection. A strain carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (sipA sopABDE2 mutant) caused the same level of fluid accumulation in bovine ligated ileal loops as a strain carrying a mutation in sipB, a SPI1 gene required for the translocation of effector proteins into host cells. A positive correlation was observed between the severity of histopathological lesions detected in the ileal mucosa and the levels of fluid accumulation induced by the different mutants. After oral infection of calves, the Salmonella serotype Typhimurium sipAsopABDE2 mutant caused only mild diarrhea and was more strongly attenuated than strains having only single mutations. These data demonstrate that SipA, SopA, SopB, SopD, and SopE2 are major virulence factors responsible for diarrhea during Salmonella serotype Typhimurium infection of calves.

Administration, Oral↗

PubMed2005

Working mechanism of immunoglobulin A1 (IgA1) protease: cleavage of IgA1 antibody to Neisseria meningitidis PorA requires de novo synthesis of IgA1 Protease.

Neisseria meningitidis secretes a protease that specifically cleaves the hinge region of immunoglobulin A1 (IgA1), releasing the effector (Fc) domain of IgA1 from the antigen binding (Fab) determinants. Theoretically, the remaining Fab fragments can block pathogen receptors or toxins and still provide protection. Here, we describe binding of V-gene-matched human IgA1 and IgA2 to PorA of strain H44/76. On live meningococci, efficient cleavage of IgA1, but not cleavage of IgA2, was observed, and up to approximately 80% of the IgA1 Fc tails were lost from the meningococcal surface within 30 min. No cleavage of IgA1 was found on an isogenic H44/76 strain lacking IgA1 protease. Furthermore, our data indicate that PorA-bound IgA1 is masked by the serogroup B polysaccharide capsule, rendering the IgA1 less accessible to degradation by secreted IgA1 protease present in the bacterial surroundings. Experiments with protein synthesis inhibitors showed that de novo production of IgA1 protease was responsible for cleavage of PorA-bound IgA1 on encapsulated bacteria. Finally, our data suggest that cleavage of IgA1 by IgA1 protease releases a significant proportion of Fab fragments from the bacterium, probably as a result of their reduced avidity compared to that of whole antibodies.

Bacterial Capsules↗

PubMed2004

Delineation of upstream signaling events in the salmonella pathogenicity island 2 transcriptional activation pathway.

Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host. Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2). SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells. A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors. While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium. In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P). Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ. In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P. As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P. Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo.

Acetate Kinase↗

PubMed2005

Transposon mutagenesis of Mb0100 at the ppe1-nrp locus in Mycobacterium bovis disrupts phthiocerol dimycocerosate (PDIM) and glycosylphenol-PDIM biosynthesis, producing an avirulent strain with vaccine properties at least equal to those of M. bovis BCG.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium bovis, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.

Animals↗

PubMed2006

Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.

Animals↗

PubMed2006

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection.

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Antigens, CD↗

PubMed2005

Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion.

BACKGROUND: Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure. RESULTS: We have performed a fresh bioinformatics analysis of the LEE. Using PSI-BLAST we have been able to identify several novel homologies between LEE-encoded and Ysc-Yop-associated proteins: Orf2/YscE, Orf5/YscL, rORF8/EscI, SepQ/YscQ, SepL/YopN-TyeA, CesD2/LcrR. In addition, we highlight homology between EspA and flagellin, and report many new homologues of the chaperone CesT. CONCLUSION: We conclude that the vast majority of LEE-encoded proteins do indeed possess homologues and that homology data can be used in combination with experimental data to make fresh functional predictions.

Amino Acid Sequence↗