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

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.

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

Infection and Immunity ◽

10.1128/iai.71.3.1352-1360.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1352-1360 ◽

Cited By ~ 19

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.

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Biogenic synthesis of silver nanoparticles from waste banana plant stems and their antibacterial activity against Escherichia coli and Staphylococcus Epidermis

International Journal of Research in Medical Sciences ◽

10.18203/2320-6012.ijrms20173947 ◽

2017 ◽

Vol 5 (9) ◽

pp. 3769 ◽

Cited By ~ 8

Author(s):

Huu Dang ◽

Derek Fawcett ◽

Gerrard Eddy Jai Poinern

Keyword(s):

Escherichia Coli ◽

Antibacterial Activity ◽

Ag Nanoparticles ◽

Inhibition Zone ◽

Gram Negative Bacteria ◽

Banana Plant ◽

Biogenic Synthesis ◽

Maximum Inhibition ◽

Staphylococcus Epidermis ◽

First Time

Background: This study for the first time presents an eco-friendly and room temperature procedure for biologically synthesizing silver (Ag) nanoparticles from waste banana plant stems.Methods: A simple and straightforward green chemistry based technique used waste banana plant stems to act as both reducing agent and capping agent to produce Ag nanoparticles, which were subsequently characterized. In addition, antibacterial studies were conducted using the Kirby-Bauer sensitivity method.Results: Advanced characterisation revealed the Ag nanoparticles had a variety of shapes including cubes, truncated triangular and hexagonal plates, and ranged in size from 70 nm up to 600 nm. The gram-negative bacteria Escherichia coli showed the maximum inhibition zone of 12 mm.Conclusions: The study has shown that waste banana plant stems can generate Ag nanoparticles with antibacterial activity against Escherichia coli and Staphylococcus epidermis.

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Enteric infections caused by non-enterotoxigenic Escherichia coli in animals: Occurence and pathogenicity mechanisms. A review

Veterinary Microbiology ◽

10.1016/0378-1135(87)90050-2 ◽

1987 ◽

Vol 14 (1) ◽

pp. 33-46 ◽

Cited By ~ 20

Author(s):

Lieve Okerman

Keyword(s):

Escherichia Coli ◽

Enterotoxigenic Escherichia Coli ◽

Enteric Infections

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Detection of the mcr-1 gene in Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains isolated from broilers

Pesquisa Veterinária Brasileira ◽

10.1590/1678-5150-pvb-5983 ◽

2020 ◽

Vol 40 (3) ◽

pp. 165-169

Author(s):

Hugo P. Lopes ◽

Gisllany A. Costa ◽

Ana C.L.Q. Pinto ◽

Leandro S. Machado ◽

Nathalie C. Cunha ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Enteropathogenic Escherichia Coli ◽

E Coli ◽

Poultry Products ◽

Drug Of Choice ◽

Bacteriological Method ◽

Enteric Infections ◽

Polymerase Chain

ABSTRACT: Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains are among the major pathotypes found in poultry and their products, which are capable of causing human enteric infections. Colistin has been claimed the drug of choice against diseases caused by multidrug-resistant Gram-negative bacteria (MDRGN) in humans. The mcr-1 gene was the first plasmidial gene that has been described to be responsible for colistin resistance and has also been detected in birds and poultry products. Our study aimed to detect the mcr-1 gene in enteropathogenic strains of E. coli in order to evaluate the resistance to colistin in broilers. The material was obtained from 240 cloacal samples and 60 broiler carcasses. The strains were isolated by the conventional bacteriological method and by the virulence genes, which characterize the enteropathogenic strains and resistance, and the samples were detected by polymerase chain reaction (PCR). Of the 213 isolated strains of E. coli, 57 (26.76%) were characterized as atypical EPEC and 35 (16.43%) as STEC. The mcr-1 gene was found in 3.5% (2/57) of the EPEC strains and 5.7% (2/35) of the STEC strains. In this study, it was possible to confirm that the mcr-1 resistance gene is already circulating in the broiler flocks studied and may be associated with the pathogenic strains.

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Composition and Antimicrobial Activity of the Essential Oil of a Wild Kiwi

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.322.160 ◽

2011 ◽

Vol 322 ◽

pp. 160-163

Author(s):

Yin Lu ◽

Hong Chen

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Antimicrobial Activity ◽

Fungal Species ◽

Gram Negative Bacteria ◽

Significant Activity ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Leaf Oil ◽

First Time

A medicinal wild kiwi in China, Actinidia valvata Dunn, has been well known for its activities against leprosy and cancers. The compositions and the antimicrobial activity of its leaf oil were reported for the first time. The oil obtained by hydrodistillation and analyzed by GC and GC-MS, was characterized by the high content of monoterpenes. Linalool (48.14%) is the major component identified, followed by 1,2-dimethyl-lindoline (7.94%), linolenic acid methylester (6.57%) and (E)-phytol (5.29%). The antimicrobial activity of the oil was evaluated against four bacterial and three fungal species. The results showed that it exhibited a mild antibacterial activity against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), a significant activity against Gram-negative bacteria (Escherichia coli), and no activity on Pseudomonas aeruginosa. The test fungi were more sensitive to the oil, with a MIC range of 0.78~1.56 μL/mL than bacteria in the range which were significantly higher from 0.78 to 25.50 μL/mL.

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

Infection and Immunity ◽

10.1128/iai.73.1.258-267.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 258-267 ◽

Cited By ~ 35

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.

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Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae

Microbiology ◽

10.1099/mic.0.079319-0 ◽

2014 ◽

Vol 160 (9) ◽

pp. 1855-1866 ◽

Cited By ~ 11

Author(s):

Shreya Dasgupta ◽

Pallabi Basu ◽

Ritesh Ranjan Pal ◽

Satyabrata Bag ◽

Rupak K. Bhadra

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Small Molecules ◽

Vibrio Cholerae ◽

Stringent Response ◽

Synthetase Activity ◽

Gram Negative Bacteria ◽

Amino Acid Residues ◽

Gram Negative

In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

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Small intestine lactobacilli growth promotion and immunomodulation in weaner pigs fed Cyberlindnera jadinii yeast high inclusion diet and exposed to enterotoxigenic Escherichia coli F4+: O149

10.1101/2021.02.11.430732 ◽

2021 ◽

Author(s):

S. Iakhno ◽

S.S. Hellestveit ◽

Ö.C.O. Umu ◽

L.T. Bogevik ◽

C.P. Åkesson ◽

...

Keyword(s):

Escherichia Coli ◽

Small Intestine ◽

Growth Promotion ◽

Enterotoxigenic Escherichia Coli ◽

Feed Additive ◽

The Novel ◽

Beta Glucans ◽

Gut Immunity ◽

Enteric Infections ◽

History Of

1AbstractEnterotoxigenic Escherichia coli (ETEC) F4+: O149 is a causative agent for the development of post-weaning diarrhoea (PWD) in pigs that contributes to production losses. Yeast cell wall components used as a feed additive can modulate gut immunity and help protect animals from enteric infections. This work investigated how a novel yeast diet with high inclusion of yeast proteins (40% of crude protein) affected the course of ETEC mediated diarrhoea in weaner piglets from a farm with or without a history of post-weaning diarrhoea. We found that immune response to F4ab ETEC infection and appetite of the animals were altered by high inclusion C. jadinii yeast. The results indicate that the novel diet can support the diseased animals either directly through the effect of yeast beta-glucans and mannans or indirectly through the promotion of small intestine lactobacilli or both.

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Attenuated Shigella flexneri 2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.68.9.4884-4892.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 4884-4892 ◽

Cited By ~ 44

Author(s):

Hilary Koprowski ◽

Myron M. Levine ◽

Richard J. Anderson ◽

Genevieve Losonsky ◽

Mariagrazia Pizza ◽

...

Keyword(s):

Escherichia Coli ◽

Vaccine Strain ◽

Guinea Pigs ◽

Oral Vaccine ◽

Shigella Flexneri ◽

Surface Expression ◽

Enterotoxigenic Escherichia Coli ◽

Secretory Iga ◽

Colonization Factor ◽

Heat Labile

ABSTRACT A multivalent live oral vaccine against both Shigellaspp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM1 binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

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