RESUMEN
The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Peste/genética , Yersinia pestis/genética , Proteínas de Unión al ADN/genética , Genes araC/genética , Humanos , Operón/genética , Peste/microbiología , Peste/prevención & control , Plásmidos/genética , Factores de Transcripción/genética , Vacunas/genética , Yersinia pestis/patogenicidadRESUMEN
Bordetella pertussis has been shown to encode regulatory RNAs, yet the posttranscriptional regulatory circuits on which they act remain to be fully elucidated. We generated mutants lacking the endonucleases RNase III and RNase E and assessed their individual impact on the B. pertussis transcriptome. Transcriptome sequencing (RNA-Seq) analysis showed differential expression of â¼25% of the B. pertussis transcriptome in each mutant, with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake (e.g., ABC transporters) and in virulence (e.g., the type III secretion system and the autotransporters vag8, tcfA, and brkA). Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions, a result that was validated by qPCR and immunoblotting for tcfA and brkA. Of note, whereas similar mutations to RNase E in Escherichia coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. We further compared our data set to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified â¼120 genes and 19 operons potentially regulated at the posttranscriptional level. Thus, our findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, we highlighted loci potentially influenced by regulatory RNAs, providing insights into the posttranscriptional regulatory networks involved in fine-tuning B. pertussis gene expression. IMPORTANCE Noncoding, regulatory RNAs in bacterial pathogens are critical components required for rapid changes in gene expression profiles. However, little is known about the role of regulatory RNAs in the growth and pathogenesis of Bordetella pertussis. To address this, mutants separately lacking ribonucleases central to regulatory RNA processing, RNase III and RNase E, were analyzed by RNA-Seq. Here, we detail the first transcriptomic analysis of the impact of altered RNA degradation in B. pertussis. Each mutant showed approximately 1,000 differentially expressed genes, with significant changes in the expression of pathways associated with metabolism, bacterial secretion, and virulence factor production. Our analysis suggests an important role for these ribonucleases during host colonization and provides insights into the breadth of posttranscriptional regulation in B. pertussis, further informing our understanding of B. pertussis pathogenesis.
Asunto(s)
Proteínas Bacterianas/genética , Bordetella pertussis/genética , Endorribonucleasas/genética , Regulación Bacteriana de la Expresión Génica/genética , Procesamiento Postranscripcional del ARN/genética , Ribonucleasa III/genética , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Bordetella pertussis/crecimiento & desarrollo , Bordetella pertussis/metabolismo , Perfilación de la Expresión Génica , Mutación , Regulón , Transcriptoma , Virulencia , Factores de Virulencia/genéticaRESUMEN
Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance. Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined. Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance. Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials.