RESUMEN
Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Lipopolisacáridos/química , Yersinia pseudotuberculosis/química , Interacciones Huésped-Patógeno/genética , Humanos , Lípido A/genética , Lípido A/inmunología , Lipopolisacáridos/genética , Lipopolisacáridos/inmunología , Estructura Molecular , Relación Estructura-Actividad , Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/patogenicidadRESUMEN
We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies.
Asunto(s)
Bacterias/crecimiento & desarrollo , Silenciador del Gen , Genes Bacterianos/fisiología , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Cloranfenicol/farmacología , Eliminación de Gen , Genoma Bacteriano , Genómica/métodosRESUMEN
A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 (racE1), glutamate racemase E2 (racE2) and comEC knock-out mutants of B. anthracis strain DeltaANR. Allelic replacements were observed at high frequencies, ranging from approximately 0.5% for racE2 up to 50% for racE1 and comEC. The system can be used for genetic validation of potential drug targets.
Asunto(s)
Bacillus anthracis/genética , Mutagénesis Insercional/métodos , Isomerasas de Aminoácido/genética , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Resistencia a la Kanamicina/genética , Metiltransferasas/genética , Datos de Secuencia Molecular , Plásmidos/genética , Racemasas y Epimerasas/genética , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.
