scholarly journals Mucosal Adjuvant Properties of Mutant LT-IIa and LT-IIb Enterotoxins That Exhibit Altered Ganglioside-Binding Activities

2005 ◽  
Vol 73 (3) ◽  
pp. 1330-1342 ◽  
Author(s):  
Hesham F. Nawar ◽  
Sergio Arce ◽  
Michael W. Russell ◽  
Terry D. Connell
Keyword(s):  
Single Point ◽  
Binding Activity ◽  
Type I ◽  
Adjuvant Activity ◽  
Adrenal Cells ◽  
E Coli ◽  
Heat Labile ◽  
Adhesin Protein ◽  
And Function ◽  
Major Ganglioside

ABSTRACT 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.

scholarly journals Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants

2006 ◽  
Vol 75 (2) ◽  
pp. 621-633 ◽  
Author(s):  
Hesham F. Nawar ◽  
Sergio Arce ◽  
Michael W. Russell ◽  
Terry D. Connell
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Binding Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Type Ii ◽  
Heat Labile ◽  
Enhanced Expression ◽  
Adhesin Protein ◽  
And Function

ABSTRACT 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.

scholarly journals Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

2016 ◽  
Vol 473 (21) ◽  
pp. 3923-3936 ◽  
Author(s):  
Dani Zalem ◽  
João P. Ribeiro ◽  
Annabelle Varrot ◽  
Michael Lebens ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Cholera Toxin ◽  
Carbohydrate Binding ◽  
Type I ◽  
Type Ii ◽  
E Coli ◽  
B Subunit ◽  
Heat Labile ◽  
B Subunits

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

Purification, characterization and induction of a C-type lectin in the freshwater planarian Dugesia japonica

2012 ◽  
Vol 7 (2) ◽  
pp. 354-361 ◽  
Author(s):  
Qiuxiang Pang ◽  
Xuemei Liu ◽  
Bosheng Zhao ◽  
Wei Wei ◽  
Xiufang Zhang ◽  
...  
Keyword(s):  
Binding Activity ◽  
Single Step ◽  
Immune Defense ◽  
Ph Optimum ◽  
Freshwater Planarian ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Sds Page ◽  
Dugesia Japonica ◽  
And Function

AbstractLectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.

scholarly journals LT-IIc, a New Member of the Type II Heat-Labile Enterotoxin Family Encoded by an Escherichia coli Strain Obtained from a Nonmammalian Host

2010 ◽  
Vol 78 (11) ◽  
pp. 4705-4713 ◽  
Author(s):  
Hesham F. Nawar ◽  
Natalie D. King-Lyons ◽  
John C. Hu ◽  
Raymond C. Pasek ◽  
Terry D. Connell
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Amino Acid Sequences ◽  
Type I ◽  
Avian Host ◽  
Type Ii ◽  
E Coli ◽  
Heat Labile ◽  
Genes Encoding ◽  
New Type

ABSTRACT Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.

scholarly journals A Heat-labile Enterotoxin B Subunit Mutant (LTB26) of Escherichia coli Enhances Mucosal Immune adjuvanticity via Increased BCR Activity

2020 ◽  
Author(s):  
Qinlin Shi ◽  
Qiujuan Wang ◽  
Sijing Chen ◽  
Sijie Gan ◽  
Tao Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Analysis Data ◽  
Binding Activity ◽  
Cell Mediated Immunity ◽  
Adjuvant Activity ◽  
B Subunit ◽  
Heat Labile ◽  
Enterotoxin B ◽  
Immune Adjuvant ◽  
Antigen Presenting

AbstractHeat-labile enterotoxin B subunit (LTB) of Escherichia coli is a potential mucosal immune adjuvant for its safety. However, the weaker adjuvant activity fails to meet the clinical requirement. Here, one of LTB mutant numbered LTB26 is constructed with enhanced mucosal immune adjuvanticity than that of LTB wild type (LTB). The transcriptome analysis data suggest that LTB26 enhances mucosal immune adjuvanticity via increased expression of BCR and MHC II+ molecular. Furthermore, LTB26 can promote both Th1 and Th2 cell mediated immunity via upregulated expression of IL-4 and IFN-γ. Flow cytometry analysis confirms that LTB26 significantly upregulates the activity of antigen presenting cells (DCs and mature macrophage) compared with LTB and LTB57 mutant. The result demonstrates that LTB26 is a potent mucosal immune adjuvant meeting clinical requirement. The GM1 ganglioside (GM1) binding activity of LTB57 is higher than that of LTB26; instead, the immune adjuvanticity of LTB57 is lower than that of LTB26. The result highlights that the immune adjuvanticity of LTB and its mutant are not positively associated with GM1 affinity, which upends decades understanding of the relationship of LTB adjuvanticity and GM1-binding affinity.

Structural similarity of ribosomes from E. coli and A. salina

1978 ◽  
Vol 36 (2) ◽  
pp. 242-243
Author(s):  
M. Boublik ◽  
R.M. Wydro ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Ribosomal Proteins ◽  
Direct Evidence ◽  
Structural Similarity ◽  
Carbon Support ◽  
Artemia Salina ◽  
Uranyl Acetate ◽  
Biological Assays ◽  
E Coli ◽  
And Function ◽  
Fine Carbon

Ribosomes are ribonucleoprotein particles necessary for processing the genetic information of mRNA into proteins. Analogy in composition and function of ribosomes from diverse species, established by biochemical and biological assays, implies their structural similarity. Direct evidence obtained by electron microscopy seems to be of increasing relevance in understanding the structure of ribosomes and the mechanism of their role in protein synthesis.The extent of the structural homology between prokaryotic and eukaryotic ribosomes has been studied on ribosomes of Escherichia coli (E.c.) and Artemia salina (A.s.). Despite the established differences in size and in the amount and proportion of ribosomal proteins and RNAs both types of ribosomes show an overall similarity. The monosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) appear in the electron micrographs as round particles with a diameter of approximately 225Å for the 70S E.c. (Fig. 1) and 260Å for the 80S A.s. monosome (Fig. 2).

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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