RESUMEN
Real-time polymerase chain reaction (PCR) assays were developed to detect Francisella tularensis (Ft), the causative agent of tularaemia in humans. Two real-time PCRs (FTT0376 and FTT0523) were designed in genetic sequences identified by the Insignia genome comparison tool (http://insignia.cbcb.umd.edu/) as being unique to pathogenic subspecies of F. tularensis. Both PCRs identified all pathogenic F. tularensis subspecies but did not cross react with avirulent Francisella philomiragia or F. tularensis ssp. novicida or other environmental bacteria. Limits of detection from DNA purified from pure culture (FTT0376 approximately 80 Ft genome equivalents (GEs) per PCR; FTT0523 approximately 20 Ft GEs per PCR;) and DNA purified from spiked blood samples (4 x 10(4) to 4 x 10(3) cfu ml(-1), both assays) were determined.
Asunto(s)
Bioensayo/métodos , Francisella tularensis/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , ADN Bacteriano/análisis , ADN Bacteriano/genética , Francisella tularensis/genética , Humanos , Ratones , Sensibilidad y EspecificidadRESUMEN
Iron is an important nutritional requirement for bacteria due to its conserved role in many essential metabolic processes. As a consequence of the lack of freely available iron in the mammalian host, bacteria upregulate a range of virulence factors during infection. Transcriptional analysis of Francisella tularensis subsp. novicida U112 grown in iron-deficient medium identified 21 genes upregulated in response to this condition, four of which were attributed to a siderophore operon. In addition, a novel iron-regulated gene, FTT0025, was identified which is part of this operon and encodes a 55 kDa hypothetical membrane protein. When grown on chrome azurol S agar, the F. tularensis subsp. novicida U112deltaFTT0025 mutant produced an increased reaction zone compared with the wild-type, suggesting that siderophore production was unaffected but that the bacteria may have a deficiency in their ability to re-sequester this iron-binding molecule. Furthermore, the deltaFTT0025 mutant was attenuated in a BALB/c mouse model of infection relative to wild-type F. tularensis subsp. novicida U112.
Asunto(s)
Proteínas Bacterianas/fisiología , Francisella tularensis/patogenicidad , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Proteínas de la Membrana/fisiología , Factores de Virulencia/fisiología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Francisella tularensis/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Supervivencia , Tularemia/microbiología , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
A gene cluster encoding enzymes involved in LPS O antigen biosynthesis was identified from the partial genome sequence of Francisella tularensis subsp. tularensis Schu S4. All of the genes within the cluster were assigned putative functions based on sequence similarity with genes from O antigen biosynthetic clusters from other bacteria. Ten pairs of overlapping primers were designed to amplify the O antigen biosynthetic cluster by PCR from nine strains of F. tularensis. Although the gene cluster was present in all strains, there was a size difference in one of the PCR products between subsp. tularensis strains and subsp. holarctica strains. LPS was purified from F. tularensis subsp. tularensis Schu S4 and the O antigen was shown by mass spectrometry to have a structure similar to that of F. tularensis subsp. holarctica strain 15. When LPS from F. tularensis subsp. tularensis Schu S4 was used to immunize mice that were then challenged with F. tularensis subsp. tularensis Schu S4, an extended time to death was observed.