RESUMEN
This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Complejo Burkholderia cepacia/fisiología , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/química , Complejo Burkholderia cepacia/genética , Caenorhabditis elegans , Evaluación Preclínica de Medicamentos , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Alineación de SecuenciaRESUMEN
The bceA gene is part of the Burkholderia cepacia IST408 exopolysaccharide (EPS) biosynthetic cluster. It encodes a 55.3-kDa bifunctional protein (type II PMI family) with phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. GMP activity is strongly dependent on the presence of Ca(2+) or Mn(2+), while PMI activity can use a broader variety of divalent cations (Ca(2+)>Mn(2+)>Mg(2+)>Co(2+)>Ni(2+)). The lack of a functional bceA gene does not affect EPS production yield in a non-polar insertion bceA mutant. The in silico search for putative bceA homologues revealed the presence of 2-5 bceA orthologues in the Burkholderia genomes available. This suggests that in B. cepacia IST408 putative bceA functional homologues may compensate the bceA mutation. However, the viscosity of aqueous solutions prepared with the EPS produced by the bceA mutant was significantly reduced compared with wild-type biopolymer and the mutant forms biofilms with a size reduced by 6-fold.
Asunto(s)
Burkholderia cepacia/enzimología , Burkholderia cepacia/genética , Manosa-6-Fosfato Isomerasa/genética , Manosa-6-Fosfato Isomerasa/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Activación Enzimática , Regiones Promotoras Genéticas/genéticaRESUMEN
Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.
Asunto(s)
Complejo Burkholderia cepacia/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biopelículas/crecimiento & desarrollo , Complejo Burkholderia cepacia/genética , Caenorhabditis elegans , Clonación Molecular , Conjugación Genética , ADN Bacteriano/genética , Expresión Génica , Genes Bacterianos , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Pigmentos Biológicos/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.
Asunto(s)
Complejo Burkholderia cepacia/patogenicidad , Caenorhabditis elegans/microbiología , Animales , Técnicas Bacteriológicas , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/aislamiento & purificación , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Microbiología Ambiental , Humanos , Modelos Animales , Mutación , Fenotipo , Especificidad de la Especie , Virulencia/genéticaRESUMEN
Burkholderia cenocepacia H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by cep, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode Caenorhabditis elegans, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the cep-regulated virulence factors required for nematode killing. Here we report the identification of a cep-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen Ralstonia solanacearum. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that B. cenocepacia H111 AidA is essential for slow killing of C. elegans but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by B. cenocepacia but also by many other strains of the Burkholderia cepacia complex.