A monoclonal antibody that targets the conserved core/lipid A region of lipopolysaccharide affects motility and reduces intestinal colonization of both classical and El Tor Vibrio cholerae biotypes

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

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Analysis of Receptor for Vibrio choleraeEl Tor Hemolysin with a Monoclonal Antibody That Recognizes Glycophorin B of Human Erythrocyte Membrane

Infection and Immunity ◽

10.1128/iai.67.10.5332-5337.1999 ◽

1999 ◽

Vol 67 (10) ◽

pp. 5332-5337 ◽

Cited By ~ 22

Author(s):

Dongyan Zhang ◽

Junko Takahashi ◽

Taiko Seno ◽

Yoshihiko Tani ◽

Takeshi Honda

Keyword(s):

Monoclonal Antibody ◽

Vibrio Cholerae ◽

Erythrocyte Membrane ◽

Human Erythrocyte ◽

Mammalian Cells ◽

Biochemical Characterization ◽

Human Erythrocytes ◽

Human Erythrocyte Membrane ◽

El Tor ◽

Glycophorin B

ABSTRACT El Tor hemolysin (ETH), a pore-forming toxin secreted byVibrio cholerae O1 biotype El Tor and most Vibrio cholerae non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes.

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Investigation of the Roles of Toxin-Coregulated Pili and Mannose-Sensitive Hemagglutinin Pili in the Pathogenesis of Vibrio cholerae O139 Infection

Infection and Immunity ◽

10.1128/iai.66.2.692-695.1998 ◽

1998 ◽

Vol 66 (2) ◽

pp. 692-695 ◽

Cited By ~ 105

Author(s):

Carol O. Tacket ◽

Ronald K. Taylor ◽

Genevieve Losonsky ◽

Yu Lim ◽

James P. Nataro ◽

...

Keyword(s):

Vibrio Cholerae ◽

Geometric Mean ◽

Deletion Mutants ◽

Intestinal Colonization ◽

Colonization Factor ◽

Vibrio Cholerae O139 ◽

El Tor ◽

Adult Volunteers

ABSTRACT In this study, adult volunteers were fed tcpA andmshA deletion mutants of V. cholerae O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 ΔmshA shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 × 105 CFU/g of stool. In contrast, only one of nine recipients of CVD 112 ΔtcpA shed vibrios in his stool (P < 0.01); during the first 24 h after inoculation, 3 × 102 CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 ΔmshA developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 ΔtcpA developed a fourfold rise in vibriocidal titer (P < 0.01). We conclude that TCP are an important colonization factor of V. cholerae O139 and probably of El Tor V. cholerae O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.

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Transcriptional profiling of Vibrio cholerae O1 following exposure to human anti- lipopolysaccharide monoclonal antibodies

Pathogens and Disease ◽

10.1093/femspd/ftaa029 ◽

2020 ◽

Vol 78 (4) ◽

Cited By ~ 2

Author(s):

Danielle E Baranova ◽

Graham G Willsey ◽

Kara J Levinson ◽

Carol Smith ◽

Joseph Wade ◽

...

Keyword(s):

Vibrio Cholerae ◽

Lipid A ◽

Transcriptional Profiling ◽

Watery Diarrhea ◽

Rna Seq ◽

Intestinal Epithelia ◽

Vibrio Cholerae O1 ◽

El Tor ◽

Upregulated Genes ◽

Late Stages

ABSTRACT Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.

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Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins

Canadian Journal of Microbiology ◽

10.1139/m90-071 ◽

1990 ◽

Vol 36 (6) ◽

pp. 409-413 ◽

Cited By ~ 5

Author(s):

Mark L. Tamplin ◽

Reema Jalali ◽

Mohammed K. Ahmed ◽

Rita R. Colwell

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Binding Site ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

B Subunit ◽

Vibrio Mimicus ◽

Vibrio Cholerae O1 ◽

El Tor ◽

Reaction Patterns

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site. Key words: cholera toxin, epitopes, monoclonal antibodies.

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Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae

Biochemical Journal ◽

10.1042/bj1670147 ◽

1977 ◽

Vol 167 (1) ◽

pp. 147-154 ◽

Cited By ~ 11

Author(s):

S Raziuddin

Keyword(s):

Fatty Acids ◽

Molecular Weight ◽

Vibrio Cholerae ◽

Lipid A ◽

Haemagglutination Inhibition ◽

Gel Filtration ◽

Free Fraction ◽

Alkaline Methanolysis ◽

El Tor

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.

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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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BINDING SPECIFICITY OF POLYMYXIN B, BPI, LALF, AND ANTI-DEEP CORE/LIPID A MONOCLONAL ANTIBODY TO LIPOPOLYSACCHARIDE PARTIAL STRUCTURES

Shock ◽

10.1097/00024382-200115020-00008 ◽

2001 ◽

Vol 15 (2) ◽

pp. 124-129 ◽

Cited By ~ 9

Author(s):

Todd A. Kellogg ◽

Victor Lazaron ◽

Karen R. Wasiluk ◽

David L. Dunn

Keyword(s):

Monoclonal Antibody ◽

Lipid A ◽

Binding Specificity ◽

Polymyxin B ◽

Partial Structures ◽

Core Lipid

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A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription

Infection and Immunity ◽

10.1128/iai.00080-20 ◽

2020 ◽

Vol 88 (5) ◽

Cited By ~ 1

Author(s):

Prosenjit Samanta ◽

Rahul Shubhra Mandal ◽

Rudra Narayan Saha ◽

Sreeja Shaw ◽

Priyanka Ghosh ◽

...

Keyword(s):

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Sensitive Strain ◽

Two Component System ◽

Content Type ◽

Vibrio Cholerae O1 ◽

Binding Cleft ◽

Culturing Conditions ◽

El Tor

ABSTRACT Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

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