Synthesis in Vibrio cholerae and secretion of hepatitis B virus antigens fused to Escherichia coli heat-labile enterotoxin subunit B

Schödel, Florian; Will, Hans; Johansson, S.; Sánchez, Jesús R.; Holmgren, Jan

doi:10.1016/0378-1119(91)90135-x

Cited by 24 publications

(15 citation statements)

References 14 publications

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“…This includes the presence of a nadC homologue upstream of pilA, as in V. cholerae and E. coli (10,67,69). The genes encoding the pilin, pilA in P. aeruginosa and tapA in A. salmonicida, are required for virulence, although a positive correlation with a pilus structure has only been shown in P. aeruginosa (4,11,37,48,57,59,61).…”

Section: Discussionmentioning

confidence: 99%

A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

2005

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Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/ N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.Vibrio vulnificus, a halophilic, estuarine bacterium, is an opportunistic human pathogen capable of causing fatal primary septicemias or necrotizing wound infections (60). Primary septicemia usually occurs in susceptible hosts following ingestion of raw seafood, mainly shellfish, which are often naturally colonized by the organism. Wound infections are usually acquired through introduction of the organism into preexisting or new wounds, often during handling of shellfish harvested from waters where the organism is present. Host risk factors that contribute to susceptibility include liver disorders such as cirrhosis and alcoholic liver disease, hematological conditions such as hemochromatosis, and compromised immune status (60).A number of individual bacterial surface factors and extracellular proteins have been implicated in the virulence and pathogenesis of V. vulnificus, including an exopolysaccharide capsule, lipopolysaccharide, metalloprotease, cytolysin, phospholipase, and siderophores (reviewed in reference 60). Construction of specific mutations in genes controlling expression of these cell-associated and extracellular factors and analysis of virulence in animal models have shown that only the exopolysaccharide capsule, the presence of a functional flagellar biogenesis system, and the siderophore to acquire iron from transferrin are essential virulence determinants (28,74,77). A determination of the in vivo role of other factors, which in general have potent in vitro activities, has remained elusive (16,49,72). Increasingly, experimental evidence suggests that virulence of V. vulnificus is multifactorial (60) and that the coordinate regulation and expression of several virulence factors are likely controlled...

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Section: Discussionmentioning

confidence: 99%

A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

2005

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“…Recombinant proteins carrying peptides inserted into CTB are more stable than those carrying peptides linked to the N or C terminus (4). Thus, whereas CTB56-63HAp could be expressed in V. cholerae, CTB::OVAp was expressed in E. coli to avoid cleavage by extracellular V. cholerae proteases known to readily destroy Cterminal peptide extensions on CTB (38).…”

Section: Methodsmentioning

confidence: 99%

Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells

et al. 2001

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Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.Cholera toxin B subunit (CTB) and the closely related Escherichia coli heat-labile enterotoxin B subunit (LTB) are highly efficient carrier molecules for the induction of mucosal antibody responses (10, 17, 27, 31) and oral tolerance (2, 17, 42). The therapeutic applications of CTB-mediated oral tolerance, as demonstrated in animal models, include the prevention and treatment of T-cell-mediated autoimmune diseases (7,44,49), immunoglobulin E (IgE)-mediated allergic reactions (37,47,55), and infection-induced pathological inflammatory conditions (32, 43). The mechanism behind CTB's or LTB's efficacy as a mucosal carrier molecule has not been fully defined but is believed to be associated with the strong binding of CTB or LTB to the GM1 receptor present on most cells in the body, including epithelial cells and leukocytes. Efficient binding to GM1 could potentially increase the uptake of antigen across the mucosa and lead to an enhanced presentation of the conjugated molecule to the immune system (17, 34). Another possibility is that CTB per se has immunomodulating properties. Indeed, it has been shown that CTB induces major histocompatibility complex (MHC) class II expression on B cells (14), enhances antigen presentation by macrophages (M) in the absence of enhanced MHC II expression (29), and blocks the development of diabetes in NOD mice through the development of regulatory cells (40).Mucosal antigen-specific antibody formation and tolerance induction share the requirement ...

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“…Previous studies have successfully produced LTB fusions in bacterial expression systems (Cardenas and Clements 1993;Pillai et al 1996;Schodel et al 1991;Sewani et al 1998) and cholera toxin B fusions in plants (Arakawa et al 1998(Arakawa et al , 2001; however, this is the first report of a plantexpressed LTB fusion. The ability to detect both components of the LTB fusion protein in processed fruit by GM 1 ganglioside-dependent ELISA indicated the addition of a linker and epitope (15 amino acids) to the carboxy terminus of LTB did not prevent LTB pentamer forma- tion.…”

Section: Discussionmentioning

confidence: 55%

Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato

et al. 2003

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Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.

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Synthesis in Vibrio cholerae and secretion of hepatitis B virus antigens fused to Escherichia coli heat-labile enterotoxin subunit B

Cited by 24 publications

References 14 publications

A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells

Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato

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