scholarly journals Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic Escherichia coli when It Is Expressed in a Shigella Live-Vector Strain

2003 ◽  
Vol 71 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
Zeev Altboum ◽  
Myron M. Levine ◽  
James E. Galen ◽  
Eileen M. Barry
Keyword(s):  
Escherichia Coli ◽  
Shigella Flexneri ◽  
Regulatory Protein ◽  
Electron Microscopic Examination ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Electron Microscopic ◽  
Bacterial Surface ◽  
Fimbrial Subunit ◽  
Mucosal Antibody

ABSTRACT The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D′, were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5α and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

scholarly journals Construction and Characterization of Bivalent Shigella flexneri 2a Vaccine Strains SC608(pCFAI) and SC608(pCFAI/LTB) That Express Antigens from Enterotoxigenic Escherichia coli

2005 ◽  
Vol 73 (1) ◽  
pp. 258-267 ◽  
Author(s):  
Ryan T. Ranallo ◽  
C. Piyumi Fonseka ◽  
Fred Cassels ◽  
Jay Srinivasan ◽  
Malabi M. Venkatesan
Keyword(s):  
Escherichia Coli ◽  
Vaccine Strain ◽  
Shigella Flexneri ◽  
Immunoblot Analysis ◽  
Enterotoxigenic Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Live Attenuated Vaccine ◽  
Fimbrial Subunit ◽  
Semialdehyde Dehydrogenase ◽  
Structural Subunit

ABSTRACT An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens. SC608 is an aspartate semialdehyde dehydrogenase (asd) derivative of SC602 (icsA iuc), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers. When administered orally at a single 104 (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis. Using asd-based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E. coli B subunit of heat-labile enterotoxin (LTB). The expression of each heterologous protein in SC608 was verified by immunoblot analysis. Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay. After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis. All immunized animals were subsequently protected against a challenge with wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test. Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively. These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities. These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.

Immunogold labeling of the bacterial surface components of shigella flexneri 2a

1995 ◽  
Vol 53 ◽  
pp. 1052-1053
Author(s):  
D.H. Chen ◽  
G.H. Wang ◽  
H.Q. Zhang ◽  
H. Li
Keyword(s):  
Outer Surface ◽  
Bacterial Cell ◽  
Shigella Flexneri ◽  
Electron Microscopic Examination ◽  
Immunogold Labeling ◽  
Electron Microscopic ◽  
Bacterial Surface ◽  
Cell Wall Binding ◽  
Shigella Flexneri 2A ◽  
Pure Cultures

Some bacterial outer membrane components are efficient protective antigens against Shigella spp. Electron microscopic immunogold labeling provides a possibility for investigating the surface exposure of antigens (1). The aim of the present study was to localise surface antigens of Shigella flexneri 2a by using an electron microscopic immunogold labeling technique.Seven monoclonal antibodies (MAbs: 2A3, 3A6,2E6,1G8,1Al and 4F1) used in this study were produced as described previously (2). Pure cultures of Shigella flaxneri 2a were grown in broth, harvested by centrifugation, prefixed and then immunolabelled by MAbs and anti-mouse IgG conjugated to colloidal gold particles (GAMG15 and GAMG5) respectively. Ultrathin sections of labelled bacteial cells were cut and viewed in a transmission electron microscope (Philips EM 400T).Electron microscopic examination of the sectioned organisms showed that gold labeling was confined to the outer surface of the bacterial cell wall. Binding of the MAb on whole bacterial cell indicated the antigens recognized by MAbs 3A6, 2E6, 4F1 and 2A3 were exposed on the outer surface of cell membrane (Fig 1, a-c), and those by MAbs 1A1 and 1G8 were not.

Production of pili on Vibrio parahaemolyticus

1988 ◽  
Vol 34 (11) ◽  
pp. 1279-1281 ◽  
Author(s):  
Takeshi Honda ◽  
Michiko Arita ◽  
Edelira Ayala ◽  
Toshio Miwatani
Keyword(s):  
Escherichia Coli ◽  
Heat Treatment ◽  
Vibrio Cholerae ◽  
Microscopic Examination ◽  
Vibrio Parahaemolyticus ◽  
Electron Microscopic Examination ◽  
Cross Reactivity ◽  
Enterotoxigenic Escherichia Coli ◽  
Electron Microscopic ◽  
Marine Agar

Electron microscopic examination showed that all strains of Vibrio parahaemolyticus examined had pili on their surface when the organism was grown on marine agar at 28 °C for 6–12 h. The pili were morphologically stable on heat treatment at 60 °C for 10 min, but both the lateral and polar flagella possessed by this organism were labile. No immunological cross-reactivity between pili of enterotoxigenic Escherichia coli and Vibrio cholerae non-01 and those of V. parahaemolyticus was observed.

scholarly journals Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charles R. Midgett ◽  
Kacey Marie Talbot ◽  
Jessica L. Day ◽  
George P. Munson ◽  
F. Jon Kull
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Family Member ◽  
Shigella Flexneri ◽  
Decanoic Acid ◽  
Enterotoxigenic Escherichia Coli ◽  
Gram Negative Bacteria ◽  
Enteric Infections ◽  
First Time ◽  
Arac Family

AbstractEnteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

scholarly journals Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pengpeng Xia ◽  
Yunping Wu ◽  
Siqi Lian ◽  
Guomei Quan ◽  
Yiting Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Symptoms ◽  
Mucosal Damage ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Epithelial ◽  
Antibody Microarray ◽  
E Coli ◽  
Fimbrial Subunit ◽  
Induced Apoptosis ◽  
Weaning Piglets

AbstractEnterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

scholarly journals Genetic Diversity of Heat-Labile Toxin Expressed by Enterotoxigenic Escherichia coli Strains Isolated from Humans

2008 ◽  
Vol 190 (7) ◽  
pp. 2400-2410 ◽  
Author(s):  
M. A. Lasaro ◽  
J. F. Rodrigues ◽  
C. Mathias-Santos ◽  
B. E. C. Guth ◽  
A. Balan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Heat Labile ◽  
Natural Diversity

ABSTRACT The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

2004 ◽  
Vol 13 (3) ◽  
pp. 295-298 ◽  
Author(s):  
Jussi J. Joensuu ◽  
Mirkka Kotiaho ◽  
Tero Riipi ◽  
Veerle Snoeck ◽  
E. Tapio Palva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transgenic Tobacco ◽  
Enterotoxigenic Escherichia Coli ◽  
Subunit Protein ◽  
Fimbrial Subunit

scholarly journals One of Two Copies of the Gene for the Activatable Shiga Toxin Type 2d in Escherichia coli O91:H21 Strain B2F1 Is Associated with an Inducible Bacteriophage

2002 ◽  
Vol 70 (8) ◽  
pp. 4282-4291 ◽  
Author(s):  
Louise D. Teel ◽  
Angela R. Melton-Celsa ◽  
Clare K. Schmitt ◽  
Alison D. O'Brien
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Shiga Toxin ◽  
Sequence Similarity ◽  
Electron Microscopic Examination ◽  
The Other ◽  
Cell Cytotoxicity ◽  
Electron Microscopic ◽  
Functional Alleles ◽  
Flanking Regions

ABSTRACT Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx 2e from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx 2d1-containing lysogen was isolated from plaques on strain DH5α that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx 2d1- and stx 2d2-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.

scholarly journals Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

2014 ◽  
Vol 197 (2) ◽  
pp. 392-403 ◽  
Author(s):  
Enrique Joffré ◽  
Astrid von Mentzer ◽  
Moataz Abd El Ghany ◽  
Numan Oezguen ◽  
Tor Savidge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fluid Secretion ◽  
Toxin Production ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Heat Labile ◽  
Virulence Potential ◽  
Colonization Factors

EnterotoxigenicEscherichia coli(ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1–enzyme-linked immunosorbent assays (GM1-ELISA) andin silicoprotein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P< 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.

Sign in / Sign up

Export Citation Format

Share Document