Bordetella pertussis-infected innate immune cells drive the anti-pertussis response of human airway epithelium.

Pertussis is a severe respiratory tract infection caused by Bordetella pertussis. This bacterium infects the ciliated epithelium of the human airways. We investigated the epithelial cell response to B. pertussis infection in primary human airway epithelium (HAE) differentiated at air-liquid interface. Infection of the HAE cells mimicked several hallmarks of B. pertussis infection such as reduced epithelial barrier integrity and abrogation of mucociliary transport. Our data suggests mild immunological activation of HAE by B. pertussis indicated by secretion of IL-6 and CXCL8 and the enrichment of genes involved in bacterial recognition and innate immune processes. We identified IL-1ß and IFNγ, present in conditioned media derived from B. pertussis-infected macrophage and NK cells, as essential immunological factors for inducing robust chemokine secretion by HAE in response to B. pertussis. In transwell migration assays, the chemokine-containing supernatants derived from this HAE induced monocyte migration. Our data suggests that the airway epithelium on its own has a limited immunological response to B. pertussis and that for a broad immune response communication with local innate immune cells is necessary. This highlights the importance of intercellular communication in the defense against B. pertussis infection and may assist in the rational design of improved pertussis vaccines.

Chicken immune response following in ovo delivery of bacterial flagellin.

In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against several viral pathogens. We investigated the immune response of chicken embryos to purified recombinant protein. In ovo delivery of Salmonella flagellin to 18-day old embryonated eggs resulted in elevated pro-inflammatory chIL-6 and chIL-8 (CXCL8-CXCLi2) cytokine transcript levels in the intestine but not in the spleen at 24 h post-injection. Analysis of the chicken Toll-like receptor (TLR) repertoire in 19-day old embryos revealed gene transcripts in intestinal and spleen tissue for most chicken TLRs, including TLR5 which recognizes Salmonella flagellin (FliC). The in ovo administration of FliC did not alter TLR transcript levels, except for an increase in intestinal chTLR15 expression. Measurement of the antibody response in sera collected at day 11 and day 21 post-hatch demonstrated high titers of FliC-specific antibodies for the animals immunized at the late-embryonic stage in contrast to the mock-treated controls. The successful in ovo immunization with purified bacterial antigen indicates that the immune system of the chicken embryo is sufficiently mature to yield a strong humoral immune response after single exposure to purified protein. This finding strengthens the basis for the development of in ovo protein-based subunit vaccines.

Trichinella spiralis-secreted products modulate DC functionality and expand regulatory T cells in vitro.

Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-ß. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.

Differential N-glycan- and protein-directed immune responses in Dictyocaulus viviparus-infected and vaccinated calves.

Calves with naturally acquired Dictyocaulus viviparus infection mount an effective immune response. In the search for protection-inducing antigens, we found that several D. viviparus third-stage larval (L3) and adult ES products carry N-glycans. Deglycosylation of the worm antigens using PNGase F resulted in reduced IgA, IgE, IgG1 and IgG2 (but not IgM) reactivities in sera of primary infected animals, suggesting that the carbohydrate moieties contained immunodominant epitopes. Challenge infection resulted in increased specific serum antibody levels against ES and L3 in the re-infected and challenge control groups. Testing of sera by enzyme-linked immunosorbent assay (ELISA) demonstrated a significant increase in IgG1 and IgE (but not IgA or IgG2) reactivity against the deglycosylated antigens in the re-infected group compared with the challenge control group. Sera from calves vaccinated with irradiated larvae showed a strong anti-N-glycan response, but no booster response against the protein backbone after challenge infection, consistent with the absence of a memory response. Together, our results suggest that D. viviparus proteins carry immunodominant N-glycan moieties that elicit a strong but short-lived immune response during infection and after vaccination, whereas the protein backbones effectively induce a memory response which results in a long-lasting, potentially protective immune response in re-infected, but not in vaccinated calves.

Vaccine potential of recombinant Ornithobacterium rhinotracheale antigens.

Ornithobacterium rhinotracheale is a pathogen involved in respiratory infection and systemic disease in poultry. Previously, eight potential vaccine candidates were identified that induced cross-protective immunity when administered to chickens as a multi-component vaccine. In this study, we analyzed the immunogenicity of these eight recombinant proteins by subunit vaccination, and characterized the different proteins and corresponding genes more thoroughly by sequencing, in vitro expression analysis, and cellular localization experiments. We found, that all genes encoding the eight antigens were highly conserved among different O. rhinotracheale serotypes, but the different antigens were not expressed by all serotypes. Cellular fractionation experiments indicated that the majority of the antigens are predominantly located in the outer membrane fraction. Vaccination of chickens with single-antigen vaccines demonstrated that the Or77 antigen was protective against serotypes that expressed Or77 in vitro, suggesting that the protein has strong potential as a vaccine antigen. Furthermore, immunization with four-component subunit vaccines indicated the existence of immunogenic synergism between the candidate vaccine antigens.

Successful selection of cross-protective vaccine candidates for Ornithobacterium rhinotracheale infection.

Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.

Passive immunization of immune-suppressed animals: chicken antibodies protect against Ornithobacterium rhinotracheale infection.

Unravelling of the protective immunity acquired during a natural infection may contribute to vaccine development. To assess the role of antibody-mediated immunity in protection against Ornithobacterium rhinotracheale infection in chickens, a novel experimental method was applied that combined immune depletion and passive transfer of immunity within the same host. Administration of cyclophosphamide (CY) to broiler chickens successfully suppressed B lymphocyte development, and therefore humoral immunity, as confirmed by histological and serological analysis. Challenge of CY-treated birds with O. rhinotracheale revealed a significantly higher pathology score in comparison to immune-competent birds that received the same bacterial challenge. Measurement of serum immunoglobulin levels of immune-competent birds revealed a positive correlation between IgA and/or IgG production and protection against infection. Passive transfer of O. rhinotracheale-specific antiserum to the immune-suppressed birds prior to pathogen challenge significantly decreased morbidity. This protective effect was not observed after administration of control sera containing similar concentrations of immunoglobulins. Together, these results provide firm evidence that chicken humoral immunity to O. rhinotracheale is a key component in protection against infection. Our data confirm that the applied immune depletion and reconstitution approach is an attractive tool to analyse the nature of the protective immune response.

Global detection and identification of Campylobacter fetus subsp. venerealis.

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is a genital infection that threatens the cattle industry. Detection and identification of Cfv are key factors in control programmes. Trade regulations should be based on scientifically and internationally accepted methods of detection and identification of Cfv. Such methods are described in the World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. A study was conducted to determine which methods are in use in OIE Member Countries and to get an overview of new or improved tests. A questionnaire was sent to OIE Member Countries, and 26 out of 166 were returned. Globally, a diversity of methods for the detection and identification of Cfv are in use. The authors conclude that there is a lack of harmonisation that may have consequences for the description of the health status of countries and may lead to disputes with respect to trade regulations.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100.

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.

Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence.

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue.

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial-substratum and bacterial-bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell-cell interactions to colonization.

Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp.

The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG-regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3',5'-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post-transcriptional regulatory mechanism. Reporter (beta-galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5' end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine-encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post-transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.

Modulation of gonococcal piliation by regulatable transcription of pilE.

The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (DeltapilE1) in which the lacIOP regulatory sequences control pilE transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level of pilE transcription, without a change in pilE sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.

Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili.

Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components.

Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.

Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis.

Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.

The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation.

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae.

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

Gonococcal invasion of epithelial cells driven by P.IA, a bacterial ion channel with GTP binding properties.

The neisserial porin P.I is a GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes and modulates host cell signaling events. Here, we report that P.I confers invasion of the bacterial pathogen Neisseria gonorrhoeae into Chang epithelial cells and that this event is controlled by GTP, as well as other phosphorus-containing compounds. Bacterial invasion was observed only for strains carrying the P.IA subtype of porin, which is typically associated with the development of disseminated neisserial disease, and did not require opacity outer membrane proteins, previously recognized as gonococcal invasins. Allelic replacement studies showed that bacterial invasiveness cotransferred with the P.IA (por1A) gene. Mutation of the P.I-associated protein Rmp did not alter the invasive properties. Cross-linking of labeled GTP to the porin revealed more efficient GTP binding to the P.IA than P.IB porin subtype. GTP binding was inhibited by an excess of unlabeled GTP, ATP, and GDP, as well as inorganic phosphate, but not by UTP or beta-glycerophosphate, fully in line with the respective invasion-inhibitory activities observed for these compounds. The P.IA-mediated cellular invasion may explain the more invasive behavior of P.IA strains in the natural infection and may broaden the basis for the development of a P.I-based gonococcal vaccine.

Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors.

Heparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp-2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N-terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin-mediated uptake of MS11-OpaA. This inhibition could be mimicked by an RGD-containing hexapeptide and by alpha 5 beta 1 integrin-specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp-2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side-chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co-receptors facilitating fibronectin-mediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking to Opa-proteoglycan complex with host cell integrin receptors.