scholarly journals Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-galactosidase and α-1,4 Galactosyltransferase enzymes in α-gal metabolism

2021 ◽  
Author(s):  
Surendra Raj Sharma ◽  
Gary Crispell ◽  
Ahmed Mohamed ◽  
Cameron Cox ◽  
Joshua Lange ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Functional Characterization ◽  
Basophil Activation Test ◽  
Amblyomma Americanum ◽  
Delayed Type Hypersensitivity ◽  
Galactose Metabolism ◽  
Significant Difference ◽  
Ige Mediated

AbstractAlpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-⍰-1,3-galactose (α-gal) injected into humans from the lone star tick (Amblyomma americanum) bite. This study aims at the functional characterization of two tick enzymes, α-D-galactosidase (ADGal) and α-1,4 galactosyltransferase (β-1,4GalT) in α-gal metabolism. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars: GlcNAc, Glc, and Xyl in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their functional role in α-gal metabolism and AGS onset. Silencing of ADGal led to the significant down regulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in ⍰-gal levels in silenced tissues. However, there was no significant difference in the level of ⍰-gal in β-1,4GalT silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal silenced salivary glands. These results provide an insight into the role of α-D galactosidase & β-1,4GalT in tick biology and the probable involvement in the onset of AGS.

scholarly journals Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-Galactosidase and β-1,4 Galactosyltransferase Enzymes in α-Gal Metabolism

2021 ◽  
Vol 11 ◽  
Author(s):  
Surendra Raj Sharma ◽  
Gary Crispell ◽  
Ahmed Mohamed ◽  
Cameron Cox ◽  
Joshua Lange ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Basophil Activation Test ◽  
Amblyomma Americanum ◽  
Delayed Type Hypersensitivity ◽  
Galactose Metabolism ◽  
Lone Star Tick ◽  
Intrinsic Factors ◽  
Significant Difference ◽  
Ige Mediated

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick’s specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and β-1,4 galactosyltransferases (β-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars—GlcNAc, Glc, and Xyl—in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in β-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and β-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS.

scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

scholarly journals Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica

2007 ◽  
Vol 6 (6) ◽  
pp. 940-948 ◽  
Author(s):  
Carrie A. Davis ◽  
Michael P. S. Brown ◽  
Upinder Singh
Keyword(s):  
Entamoeba Histolytica ◽  
Splice Site ◽  
Expressed Sequence Tag ◽  
Functional Characterization ◽  
Molecular Evidence ◽  
Spliceosomal Introns ◽  
Accurate Expression ◽  
Eukaryotic Organisms

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

scholarly journals Identification of an isoflavonoid transporter required for the nodule establishment of the Rhizobium-Fabaceae symbiotic interaction

2021 ◽  
Author(s):  
Wanda Biala-Leonhard ◽  
Laura Zanin ◽  
Stefano Gottardi ◽  
Rita de Brito Francisco ◽  
Silvia Venuti ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Crop Productivity ◽  
Signaling Cascades ◽  
Nutritional Deficiencies ◽  
White Lupin ◽  
Symbiotic Interaction ◽  
Phosphate Availability ◽  
Rhizobial Symbiosis

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by e.g., forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids, that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

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