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.

scholarly journals Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries

2018 ◽  
Vol 1 (5) ◽  
pp. e201800148 ◽  
Author(s):  
Britta Seip ◽  
Guénaël Sacheau ◽  
Denis Dupuy ◽  
C Axel Innis
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Throughput ◽  
Quantitative Measure ◽  
Ribosome Profiling ◽  
Coding Region ◽  
Versatile Tool

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-boundEscherichia coliribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational 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 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 Primary Structure of Human Platelet Factor 4.

1977 ◽  
Author(s):  
F.J. Morgan ◽  
G.S. Begg ◽  
C.N. Chesterman
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Information ◽  
Human Platelet ◽  
Specific Protein ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Tryptic Peptides

The amino acid sequence of human platelet factor 4 (PF4) has been studied. PF4 is a platelet specific protein with antiheparin activity, released from platelets as a proteoglycan complex, whose measurement may provide an important index of platelet activation both in vivo and in vitro. These studies were undertaken to characterize fully the PF4 molecule. PF4 is a stable tetramer, composed of identical subunits, each with a molecular weight based on the sequence studies of approx. 7,770. Each PF4 subunit contains 69 amino acids, including 4 half-cystine (# 10, 12, 36, 37), one tyrosine (# 59), 3 arginine and 8 lysine, but no methionine, phenylalanine or tryptophan residues. The basic residues are predominantly in the C-terminal region. The tryptic peptides were aligned after studies which included tryptic digestion of citraconylated RCM-PF4, and automated Edman degradation of RCM-PF4 and citraconylated tryptic peptides. No glycopeptides were detected. This structural information should enable clear distinction to be made between PF4 and other platelet proteins such as β thromboglobulin. The provisional amino acid sequence of each subunit is:Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Cys-Pro-Thr-Ala-Gln-Ile-Leu-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Pro-Leu-Asp-Leu-Gln-Ala-Tyr-Leu-Lys-Ile-Lys(Lys, Lys, Ser, Glx, Leu, Leu)

scholarly journals Bacteriostasis of Escherichia coli by milk. VI. The in-vitro bacteriostatic property of Gambian mothers' breast milk in relation to the in-vivo protection of their infants against diarrhoeal disease

1980 ◽  
Vol 85 (3) ◽  
pp. 405-413 ◽  
Author(s):  
M. G. M. Rowland ◽  
T. J. Cole ◽  
Maura Tully ◽  
Jean M. Dolby ◽  
Pauline Honour
Keyword(s):  
Escherichia Coli ◽  
Protective Action ◽  
Age Groups ◽  
Field Evaluation ◽  
Diarrhoeal Disease ◽  
Gastrointestinal Diseases ◽  
Bacteriostatic Activity ◽  
E Coli

SUMMARYA one-year field-study has been carried out in a diarrhoea-endemic area in West Africa to determine the relationship between the bacteriostatic activity of fresh human milk for Escherichia coli in vitro and freedom from diarrhoea of the infant recipients of the milk. The specific contribution of E. coli gastroenteritis to gastrointestinal diseases of infants in general is not known, nor is its particular role in the Gambian infants studied. During the study period, however, both enteropathogenic and toxigenic strains of E. coli were isolated.The incidence of diarrhoea in Gambian infants of seven age-groups from 2 days to 12 months was not significantly correlated with the bacteriostatic activity of milk. This was due rather to absence of diarrhoea in babies fed on low-activity milk than illness in those receiving highly bacteriostatic milk. Indeed, very active milk appeared to protect recipients almost completely, including seven babies of over 3 months of age, five of them during the rainy season, when the risk was high. Babies receiving lower-activity milk experienced more diarrhoea. In a situation where diarrhoeal disease is multifactorial, field evaluation of the protective action by one antibacterial property of milk is difficult. A better understanding of in vivo protection is important, and the factors which have to be taken into account are discussed.

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.

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