Biomedical subjects

Terry D Connell

Publications and source records attributed to Terry D Connell.

14 recordsLinked to original sources

PubMed2007

Ganglioside GD1a is an essential coreceptor for Toll-like receptor 2 signaling in response to the B subunit of type IIb enterotoxin.

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)). Both LT-IIb-B(5) and a GD1a binding-defective mutant (LT-IIb-B(5)(T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B(5) induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B(5)-induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.

Animals↗

PubMed2007

In vitro induction of immunoglobulin A (IgA)- and IgM-secreting plasma blasts by cholera toxin depends on T-cell help and is mediated by CD154 up-regulation and inhibition of gamma interferon synthesis.

Cholera toxin (CT) and the type II heat-labile enterotoxins (LT-IIa and LT-IIb) are potent immunological adjuvants which are hypothesized to enhance the production of antibody (Ab)-secreting cells, although their mechanisms of action are not fully understood. The treatment of splenic cells with concanavalin A (ConA) plus CT enhanced the production of immunoglobulin A (IgA) and IgM by dividing cells that expressed high levels of major histocompatibility complex class II (MHC-II), CD19, and CD138 and low levels of B220 a phenotype characteristic of plasma blasts. LT-IIa or LT-IIb moderately enhanced IgA and IgM production without enhancing plasma blast differentiation. CT up-regulated CD25, CD69, CD80, CD86, and MHC-II in isolated B cells but failed to induce proliferation or differentiation. The treatment of unfractionated splenic cells with ConA plus CT induced B-cell proliferation and differentiation, but the elimination of CD4(+) T cells inhibited this effect. CT treatment of ConA-activated CD4(+) T cells up-regulated CD134 and CD154, whereas the blockage of CD40-CD154 interactions inhibited the induction of plasma blasts and Ig synthesis. The treatment of unfractionated splenic cells with CT, LT-IIa, or LT-IIb enhanced the production of interleukin-6 (IL-6) and IL-10, whereas the production of gamma interferon was inhibited in both CD4(+) and CD8(+) T cells mostly by CT. Thus, major regulatory effects of CT on lymphocytes are likely exerted early during the induction of immune responses when B and T cells initially encounter antigen. Neither LT-IIa or LT-IIb had these effects, indicating that type II enterotoxins augment Ab responses by other mechanisms.

Animals↗

PubMed2006

Mutants of type II heat-labile enterotoxin LT-IIa with altered ganglioside-binding activities and diminished toxicity are potent mucosal adjuvants.

The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.

Adhesins, Bacterial↗

PubMed2006

Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction.

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.

Animals↗

PubMed2005

Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract.

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

ADP Ribose Transferases↗

PubMed2005

Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities.

LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

Adjuvants, Immunologic↗

PubMed2005

Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins.

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1beta (IL-1beta), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-kappaB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities.

Animals↗

PubMed2005

Transcriptional control of the rhuIR-bhuRSTUV heme acquisition locus in Bordetella avium.

Iron (Fe) is an essential nutrient for most bacterial pathogens. In these organisms, a variety of regulatory systems that respond to specific Fe complexes found within their vertebrate hosts have evolved. In Bordetella avium, the heme utilization locus encoded by rhuIR-bhuRSTUV mediates efficient acquisition of Fe from heme and hemoproteins. Control of bhuRSTUV expression is promulgated at two levels. When Fe is abundant, expression is repressed in a Fur-dependent manner which is partially relieved when Fe is limiting. In the presence of heme or hemoproteins, expression of the bhuRSTUV operon is induced via a three-component signal transduction cascade composed of RhuI, RhuR, and BhuR. Herein, we report the identification of two promoters (PrhuI and PbhuR) that control expression of the rhuIR-bhuRSTUV cluster. Primer extension analysis identified the transcriptional start site of PrhuI within a putative Fur box. Transcriptional initiation of PbhuR mapped within the rhuR-bhuR intergenic region. Maximal transcription from PbhuR required Fe-limiting conditions, the presence of heme (or hemoglobin), and rhuI; however, analysis of transcripts produced from the rhuIR-bhuRSTUV locus revealed a pattern of low-level bhuR transcription in the absence of heme which originated from both PbhuR and PrhuI. Transcription from PrhuI was repressed by Fe in the presence of fur and somewhat enhanced by the addition of hemin to Fe-limited media. The nature of this hemin-associated PrhuI stimulation was rhuI independent and therefore not induced by heme via the BhuR-RhuR-RhuI signal cascade. Fe also repressed transcription from PbhuR in a fur-dependent manner; however, activation from this promoter, in the presence or absence of heme, did not occur without rhuI.

Animals↗

PubMed2005

Differential binding of Escherichia coli enterotoxins LT-IIa and LT-IIb and of cholera toxin elicits differences in apoptosis, proliferation, and activation of lymphoid cells.

Cholera toxin (CT), LT-IIa, and LT-IIb are potent adjuvants which induce distinct T-helper (Th)-cell cytokine profiles and immunoglobulin G (IgG) subclass and IgA antibody responses. To determine if the distinct immune regulatory effects observed for LT-IIa, LT-IIb, and CT are elicited by binding of the enterotoxins to their cognate ganglioside receptors, the lineages of lymphoid cells that interact with the three enterotoxins and their effects on various lymphocyte responses in vitro were evaluated. Binding patterns of LT-IIa, LT-IIb, and CT to several lymphoid cell populations were distinctive for each enterotoxin. LT-IIa and CT, but not LT-IIb, induced apoptosis in CD8(+) T cells. LT-IIa(T34I), a mutant with no detectable binding to gangliosides, did not induce apoptosis. Blockade of GM(1) on the surface of CD8(+) T cells by LT-IIa(T14I), a mutant that binds only to GM(1) but does not induce apoptosis, did not inhibit induction of apoptosis by LT-IIa. Mitogen-induced proliferation of CD8(+) T cells was abrogated by treatment with CT, while resting CD8(+) T cells which were sensitive to LT-IIa-induced apoptosis became more resistant to apoptosis after mitogen activation. Exposure to CT, but not to LT-IIa or LT-IIb, inhibited mitogen-driven CD4(+) T-cell proliferation and expression of CD25 and CD69. In mitogen-stimulated B cells, CT, but not LT-IIa or LT-IIb, enhanced expression levels of CD86, while only CT induced B-cell differentiation into plasma cells. Thus, LT-IIa, LT-IIb, and CT exhibit distinguishable immunomodulatory properties which are likely dependent upon their capacities to recognize different ganglioside receptors on lymphocytes.

Animals↗

PubMed2004

The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells.

The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor alpha [TNF-alpha]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.

Bacterial Toxins↗

PubMed2004

RhuR, an extracytoplasmic function sigma factor activator, is essential for heme-dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella avium.

Genes involved in iron (Fe) acquisition often are regulated in response to the local availability of Fe. In many bacteria, Fe-dependent responsiveness is mediated by Fur, a global Fe-dependent transcriptional repressor. Tighter regulatory control of Fur-responsive genes is afforded by incorporating additional regulators into Fur-dependent regulatory cascades. RhuI, a Fur-dependent extracytoplasmic function sigma factor of Bordetella avium, in response to the dual stimulation of Fe starvation and the presence of heme (or hemoproteins), regulates P(bhuR), a heme-responsive promoter which directs expression of the bhuRSTUV heme utilization operon. While BhuR, the outer membrane heme receptor, and RhuI have been shown to be indispensable for heme-dependent activation of P(bhuR), collateral components of the regulatory cascade have not been described. In this investigation, RhuR, an integral cytoplasmic membrane protein with homology to anti-sigma factors, is shown to be an essential activator of P(bhuR) expression. The functional domain of RhuR required for heme-dependent activation of P(bhuR) expression was mapped to the N-terminal 97 amino acids of the protein by use of a chimeric RhuR-BlaM fusion. Expression of the chimera in a rhuR mutant rendered P(bhuR) constitutive, thereby decoupling the promoter from heme dependency. Growth studies confirmed that B. avium requires RhuR for optimal utilization of hemoglobin, but not hemin, as a sole source of nutrient Fe. These data imply that B. avium expresses, in addition to the BhuR heme/hemoprotein utilization system, an alternative RhuR-independent heme utilization mechanism. A model is proposed in which RhuR is the functional bridge between BhuR and RhuI in a heme-dependent regulatory cascade.

Amino Acid Sequence↗

PubMed2002

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide inhibit dendritic growth in cultured sympathetic neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are related neuropeptides that are released by the preganglionic sympathetic axons. These peptides have previously been implicated in the regulation of sympathetic neurotransmitter metabolism and cell survival in postganglionic sympathetic neurons. In this study we consider the possibility that PACAP and VIP also affect the morphological development of these neurons. Postganglionic rat sympathetic neurons formed extensive dendritic arbors after exposure to bone morphogenetic protein-7 (BMP-7) in vitro. PACAP and VIP reduced BMP-7-induced dendritic growth by approximately 70-90%, and this suppression was maintained for 3 weeks. However, neither PACAP nor VIP affected axonal growth or cell survival. The actions of PACAP and VIP appear to be mediated by PAC1 receptors because their effects were suppressed by an antagonist that binds to PAC1 and VPAC2 receptors (PACAP6-38), but not by an antagonist that binds to the VPAC1 and VPAC2 receptors. Moreover, exposure to PACAP and VIP caused phosphorylation and nuclear translocation of cAMP response element-binding protein, and agents that increase the intracellular concentration of cAMP mimicked the PACAP-induced inhibition of dendritic growth. These data suggest that peptides released by preganglionic nerves modulate dendritic growth in sympathetic neurons by a cAMP-dependent mechanism.

Active Transport, Cell Nucleus↗

PubMed2002

BhuR, a virulence-associated outer membrane protein of Bordetella avium, is required for the acquisition of iron from heme and hemoproteins.

Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

Amino Acid Sequence↗

PubMed2002

The extracellular transport signal of the Vibrio cholerae endochitinase (ChiA) is a structural motif located between amino acids 75 and 555.

ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kan(r)), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

Amino Acid Motifs↗