RESUMEN
Bordetella bronchiseptica PagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPa is required for the modification of lipid A with palmitate. While PagPBB transfers a single palmitate to the lipid A C-3' position, PagPBPa transfers palmitates to the lipid A C-2 and C-3' positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagPBPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.
Asunto(s)
Aciltransferasas/metabolismo , Bordetella parapertussis/enzimología , Lípido A/metabolismo , Palmitatos/metabolismo , Aciltransferasas/genética , Bordetella parapertussis/química , Bordetella parapertussis/genética , Análisis Mutacional de ADN , Lípido A/química , Espectrometría de MasasRESUMEN
The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 A resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal alpha-helix. The structure of the first subdomain (residues 1-117), which consists mostly of beta-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.
Asunto(s)
Escherichia coli/enzimología , Fragmentos de Péptidos/química , Proteasa La/química , Bordetella parapertussis/enzimología , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
A narrow-spectrum clavulanic acid-inhibited class A beta-lactamase, BOR-1, was identified in a Bordetella bronchiseptica clinical isolate. It shared 45% amino acid identity with L-2 from Stenotrophomonas maltophilia. An identical beta-lactamase gene was found in B. bronchiseptica and Bordetella parapertussis reference strains that may contribute only in part to their resistance phenotype.