Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis

ABSTRACT Although Acinetobacter baumannii has emerged as a significant cause of nosocomial infections worldwide, there have been few investigations describing the factors important for A. baumannii persistence and pathogenesis. This paper describes the first reported identification of a glycosyltransferase, LpsB, involved in lipopolysaccharide (LPS) biosynthesis in A. baumannii. Mutational, structural, and complementation analyses indicated that LpsB is a core oligosaccharide glycosyl transferase. Using a genetic approach, lpsB was compared with the lpsB homologues of several A. baumannii strains. These analyses indicated that LpsB is highly conserved among A. baumannii isolates. Furthermore, we developed a monoclonal antibody, monoclonal antibody 13C11, which reacts to an LPS core epitope expressed by approximately one-third of the A. baumannii clinical isolates evaluated to date. Previous studies describing the heterogeneity of A. baumannii LPS were limited primarily to structural analyses; therefore, studies evaluating the correlation between these surface glycolipids and pathogenesis were warranted. Our data from an evaluation of LpsB mutant 307::TN17, which expresses a deeply truncated LPS glycoform consisting of only two 3-deoxy-d-manno-octulosonic acid residues and lipid A, suggest that A. baumannii LPS is important for resistance to normal human serum and confers a competitive advantage for survival in vivo. These results have important implications for the role of LPS in A. baumannii infections.

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Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

Infection and Immunity ◽

10.1128/iai.00140-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2370-2376 ◽

Cited By ~ 14

Author(s):

Louise M. Temple ◽

David M. Miyamoto ◽

Manju Mehta ◽

Christian M. Capitini ◽

Stephen Von Stetina ◽

...

Keyword(s):

Whooping Cough ◽

Bioinformatic Analysis ◽

Tracheal Ring ◽

Tracheal Cell ◽

Bordetella Avium ◽

Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

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Roles of 3-Deoxy-d-manno-2-Octulosonic Acid Transferase from Moraxella catarrhalis in Lipooligosaccharide Biosynthesis and Virulence

Infection and Immunity ◽

10.1128/iai.73.7.4222-4230.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4222-4230 ◽

Cited By ~ 24

Author(s):

Daxin Peng ◽

Biswa P. Choudhury ◽

Ronald S. Petralia ◽

Russell W. Carlson ◽

Xin-Xing Gu

Keyword(s):

Moraxella Catarrhalis ◽

Lipid A ◽

Therapeutic Interventions ◽

Core Oligosaccharide ◽

Gene Encoding ◽

Oligosaccharide Moiety ◽

Normal Human ◽

Human Infections

ABSTRACT Lipooligosaccharide (LOS), a major outer membrane component of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of human infections caused by the organism. However, information about the roles of the oligosaccharide chain from LOS in bacterial infection remains limited. Here, a kdtA gene encoding 3-deoxy-d-manno-2-octulosonic acid (Kdo) transferase, which is responsible for adding Kdo residues to the lipid A portion of the LOS, was identified by transposon mutagenesis and construction of an isogenic kdtA mutant in strain O35E. The resulting O35EkdtA mutant produced only lipid A without any core oligosaccharide, and it was viable. Physicochemical and biological analysis revealed that the mutant was susceptible to hydrophobic reagents and a hydrophilic glycopeptide and was sensitive to bactericidal activity of normal human serum. Importantly, the mutant showed decreased toxicity by the Limulus amebocyte lysate assay, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. These data suggest that the oligosaccharide moiety of the LOS is important for the biological activity of the LOS and the virulence capability of the bacteria in vitro and in vivo. This study may bring new insights into novel vaccines or therapeutic interventions against M. catarrhalis infections.

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Identification and Characterization of a Monoclonal Antibody that Neutralizes Ricin Toxicity in Vitro and in Vivo

Hybridoma ◽

10.1089/hyb.1994.13.417 ◽

1994 ◽

Vol 13 (5) ◽

pp. 417-421 ◽

Cited By ~ 71

Author(s):

PAUL V. LEMLEY ◽

PETER AMANATIDES ◽

D. CRAIG WRIGHT

Keyword(s):

Monoclonal Antibody ◽

Toxicity In Vitro ◽

Identification And Characterization

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Characterization of a novel protective monoclonal antibody that recognizes an epitope common to Vibrio cholerae Ogawa and Inaba serotypes

Microbiology ◽

10.1099/mic.0.025726-0 ◽

2009 ◽

Vol 155 (7) ◽

pp. 2353-2364 ◽

Cited By ~ 13

Author(s):

Madushini N. Dharmasena ◽

Shelly J. Krebs ◽

Ronald K. Taylor

Keyword(s):

Monoclonal Antibody ◽

Phage Display ◽

Vibrio Cholerae ◽

Lipid A ◽

Passive Immunization ◽

Peptide Mimics ◽

Peptide Mimic ◽

Phage Display Libraries ◽

Identification And Characterization

A novel protective monoclonal antibody (mAb) that recognizes a lipopolysaccharide (LPS) epitope common between serotypes Ogawa and Inaba of the O1 serogroup of Vibrio cholerae was characterized and the potential to develop peptide mimics of this protective LPS epitope was investigated. mAb 72.1 recognizes both Ogawa and Inaba LPS and it is vibriocidal and protective in passive immunization against infection by strains of both serotypes. The cDNA-derived amino acid sequence of mAb 72.1 is closely related to the previously characterized mAb ZAC-3, which is thought to recognize an epitope in the lipid A core region of O1 LPS. In an attempt to develop a peptide mimic-based vaccine against V. cholerae, phage display libraries were screened with mAb 72.1 and 11 peptide mimics were identified. Remarkably, all of the peptide sequences identified from linear phage display libraries contained two cysteine residues, suggesting that mAb 72.1 preferentially binds to peptides constrained with a disulphide bond. One of the peptide mimics was immunologically characterized. Although immunization of mice with this peptide mimic conjugated to KLH elicited antibodies against the peptide itself, these antibodies did not cross-react with Ogawa or Inaba LPS. Effectiveness of a peptide mimic as a vaccine may depend on how well the peptide can mimic the carbohydrate interactions when binding to the anti-carbohydrate antibody. Thus, investigating how peptides and LPS bind to mAb 72.1 may be useful in improving current peptide mimics or designing more effective peptide mimics. Identification and characterization of novel protective anti-LPS antibodies may be useful in studying protective epitopes of LPS, which may help develop LPS-based therapeutics against V. cholerae.

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The thermostable 4,6-α-glucanotransferase of Bacillus coagulans DSM 1 synthesizes isomaltooligosaccharides

Amylase ◽

10.1515/amylase-2021-0002 ◽

2021 ◽

Vol 5 (1) ◽

pp. 13-22

Author(s):

Gang Xiang ◽

Piet L. Buwalda ◽

Marc J.E.C van der Maarel ◽

Hans Leemhuis

Keyword(s):

Bacillus Coagulans ◽

Glycoside Hydrolase Family ◽

Rat Intestine ◽

Glycaemic Response ◽

Food Ingredient ◽

Starch Conversion ◽

Identification And Characterization ◽

Hydrolase Family

Abstract The 4,6-α-glucanotransferases of the glycoside hydrolase family 70 can convert starch into isomaltooligosaccharides (IMOs). However, no thermostable 4,6-α-glucanotransferases have been reported to date, limiting their applicability in the starch conversion industry. Here we report the identification and characterization of a thermostable 4,6-α-glucanotransferase from Bacillus coagulans DSM 1. The gene was cloned and the recombinant protein, called BcGtfC, was produced in Escherichia coli. BcGtfC is stable up to 66 °C in the presence of substrate. It converts debranched starch into an IMO product with a high percentage of α-1,6-glycosidic linkages and a relatively high molecular weight compared to commercially available IMOs. Importantly, the product is only partly and very slowly digested by rat intestine powder, suggesting that the IMO will provide a low glycaemic response in vivo when applied as food ingredient. Thus, BcGtfC is a thermostable 4,6-α-glucanotransferase suitable for the industrial production of slowly digestible IMOs from starch.

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