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.

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

scholarly journals Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

1997 ◽  
Vol 139 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter Mundel ◽  
Hans W. Heid ◽  
Thomas M. Mundel ◽  
Meike Krüger ◽  
Jochen Reiser ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cultured Cells ◽  
Rat Kidney ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Globular Domain ◽  
Postnatal Maturation ◽  
Human And Mouse

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

scholarly journals In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

Vaccine ◽  
2009 ◽  
Vol 27 (32) ◽  
pp. 4302-4308 ◽  
Author(s):  
Shuang Liang ◽  
Kavita B. Hosur ◽  
Hesham F. Nawar ◽  
Michael W. Russell ◽  
Terry D. Connell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
B Subunit ◽  
Heat Labile

scholarly journals Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

Adsorption of Cholera and Heat-Labile Escherichia coli Enterotoxins by Various Adsorbents: an In Vitro Study

1997 ◽  
Vol 60 (4) ◽  
pp. 358-362 ◽  
Author(s):  
J. RAMU ◽  
K. CLARK ◽  
G. N. WOODE ◽  
A. B. SARR ◽  
T. D. PHILLIPS
Keyword(s):  
Escherichia Coli ◽  
Tumor Cell Line ◽  
In Vitro Study ◽  
Bentonite Clay ◽  
Published Data ◽  
Sodium Bentonite ◽  
Heat Labile ◽  
Inorganic Adsorbents

A variety of common inorganic adsorbents representing aluminas, zeolites, phyllosilicate clays, silica, and carbon were compared for their abilities to adsorb cholera toxin (CT) and heat-labile (LT)Escherichia coli enterotoxin. An appropriate assay system for the enterotoxins was developed using the Y-1 mouse-adrenal-tumor cell line, End points were determined by counting the number of rounded (cytotonic) cells at the relevant dilution. The adsorption varied between 177.0 × 106 and 109.6 × 102 CYTU (cytotonic titer unit) for CT with charcoal and boehmite respectively, and between 60.7 × 104 and 180.4 × 101 CYTU for LT with charcoal and boehmite respectively. Several of the other materials adsorbed CT and LT well, particularly attapulgite and sodium bentonite. The tightness of CT and LT binding to sodium bentonite and charcoal was determined by washing the adsorbent-enterotoxin pellets. Both toxins were strongly adsorbed, with dissociation of only 46.3 × 10° CYTU (<0.01 %) of the bound CT from sodium bentonite and 18.0× 101 CYTU (0.06%) of the bound LT from charcoal. The clay and charcoal pellets were assayed for their cytotonicity. Most of the activity of the adsorbed enterotoxins was lost: 93.1 and 89.6% for CT with sodium bentonite and charcoal, respectively, and 93.8 and 85.9% for LT with sodium bentonite and charcoal, respectively. The effect of dietary protein (casein) in enterotoxin adsorption by clay was also investigated. One percent casein (when adsorbed to sodium bentonite clay) completely blocked the adsorption of CT. When this protein-clay complex was treated with enzymes present in pancreatin, the digestive effect on the casein was sufficient to permit the adsorption of 137.6 × 101 CYTU of CT, although most of the blocking effect of casein remained. Further in vitro studies are needed to model the stomach, pancreatic, and intestinal digestive systems for determining if dietary proteins can block CT adsorption by clay in vivo. These results extend and support previously published data, obtained experimentally in rabbit and rat intestinal loops and from studies of children suffering spontaneous diarrhea, on the beneficial role of clays and other inorganic adsorbents in controlling enterotoxin activity.

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.

scholarly journals Heat-Labile Enterotoxin Promotes Escherichia coli Adherence to Intestinal Epithelial Cells

2008 ◽  
Vol 191 (1) ◽  
pp. 178-186 ◽  
Author(s):  
Amber M. Johnson ◽  
Radhey S. Kaushik ◽  
David H. Francis ◽  
James M. Fleckenstein ◽  
Philip R. Hardwidge
Keyword(s):  
Escherichia Coli ◽  
Surface Expression ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Host Interactions ◽  
Heat Labile ◽  
Vitro Model ◽  
Bacterial Sensing

ABSTRACT Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3′,5′-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.

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