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1.
Infect Immun ; 84(5): 1642-1649, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26975993

RESUMO

CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities; the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms.


Assuntos
Aderência Bacteriana , Escherichia coli Enterotoxigênica/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/fisiologia , Gangliosídeo G(M1)/metabolismo , Animais , Células CACO-2 , Células Epiteliais/microbiologia , Eritrócitos/microbiologia , Humanos , Ligação Proteica , Coelhos
2.
Adv Appl Microbiol ; 90: 155-97, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25596032

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a major cause of life-threatening diarrheal disease around the world. The major aspects of ETEC virulence are colonization of the small intestine and the secretion of enterotoxins which elicit diarrhea. Intestinal colonization is mediated, in part, by adhesins displayed on the bacterial cell surface. As colonization of the intestine is the critical first step in the establishment of an infection, it represents a potential point of intervention for the prevention of infections. Therefore, colonization factors (CFs) have been important subjects of research in the field of ETEC virulence. Research in this field has revealed that ETEC possesses a large array of serologically distinct CFs that differ in composition, structure, and function. Most ETEC CFs are pili (fimbriae) or related fibrous structures, while other adhesins are simple outer membrane proteins lacking any macromolecular structure. This chapter reviews the genetics, structure, function, and regulation of ETEC CFs and how such studies have contributed to our understanding of ETEC virulence and opened up potential opportunities for the development of preventive and therapeutic interventions.


Assuntos
Escherichia coli Enterotoxigênica/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Fatores de Virulência/metabolismo , Animais , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/isolamento & purificação , Escherichia coli Enterotoxigênica/patogenicidade , Proteínas de Escherichia coli/genética , Humanos , Virulência , Fatores de Virulência/genética
3.
Genome Announc ; 2(2)2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24723709

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease around the globe, causing an estimated 380,000 deaths annually. The disease is caused by a wide variety of strains. Here, we report the genome sequence of ETEC strain B2C, which was isolated from an American soldier in Vietnam.

4.
Biochemistry ; 52(31): 5265-79, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23841450

RESUMO

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein-protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Δhop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.


Assuntos
Pareamento Cromossômico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Motivos de Aminoácidos , DNA/química , DNA/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/química , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
5.
Protein Expr Purif ; 72(1): 42-7, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20347988

RESUMO

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively.


Assuntos
Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/genética , DNA/metabolismo , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para Cima
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