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1.
Biochem Biophys Rep ; 8: 55-61, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28955942

RESUMO

Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.

2.
FEBS J ; 282(11): 2097-114, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25845800

RESUMO

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.


Assuntos
Aminoaciltransferases/fisiologia , Proteínas de Bactérias/fisiologia , Cisteína Endopeptidases/fisiologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/química , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Humanos , Ligação Proteica , Conformação Proteica , Especificidade da Espécie , Especificidade por Substrato
3.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 7): 1983-93, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25004975

RESUMO

Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Šresolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33-497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.


Assuntos
Clostridioides difficile/química , Cisteína Endopeptidases/química , Cisteína Proteases/química , Lectinas/química , Sequência de Aminoácidos , Primers do DNA , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
4.
Biochem J ; 436(3): 517-26, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21615333

RESUMO

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Enterotoxinas , ADP Ribose Transferases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Enterotoxinas/química , Enterotoxinas/metabolismo , Glucosiltransferases/química , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X , Proteínas rho de Ligação ao GTP/metabolismo
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