RESUMO
Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Bactérias/uso terapêutico , Proteínas de Membrana/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/uso terapêutico , Proteínas de Bactérias/genética , Vacinas Bacterianas , Ligação Competitiva , Método Duplo-Cego , Proteínas de Fímbrias , Proteínas de Membrana/genética , Proteínas de Membrana/uso terapêutico , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Taxa de SobrevidaRESUMO
In this study, we cloned and sequenced the Helicobacter pylori genes encoding fumarate reductase (FRD). H. pylori frdA, frdB and frdC specify polypeptides of 715, 245 and 254 aa, respectively. The deduced aa sequences of FrdA and FrdB are highly homologous to those of the corresponding subunits of Wolinella succinogenes FRD and also exhibit a significant sequence identity with other bacterial FRD and succinate dehydrogenase subunits A and B. However, H. pylori FrdC shares a striking degree of sequence identity only with W. succinogenes FrdC, which is a cytochrome b with two haem groups. The products encoded by H. pylori frdA, frdB and frdC were overproduced in maxicells and H. pylori FrdA was characterized using an anti-E. coli FrdA serum. H. pylori FRD activity, which was measured as fumarate-dependent benzyl viologen oxidation, is membrane-associated. Inactivation of frdA led to the loss of such activity and the mutant H. pylori cells were delayed (approx. 10-20 h behind their parent cells) in entering the mid-log phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H. pylori cells in vitro. H. pylori FRD contains three subunits, of which FrdA and FrdB appear to form the catalytic dimer, whereas FrdC serves as a membrane anchor.