Project description:Objectives: To determine the transcripts that are differentially expressed in a hfq mutant. Hfq is an RNA chaperone that mediates many interactions between regultory RNAs and their mRNA targets. Analysis of the transcriptomes of the Pasteurella multocida wild-type strain and the Pasteurella multocida hfq mutant will allow for identification of genes controlled by hfq and the sRNAs with which it interacts. Methods: RNA sequencing was employed to determine the transcriptomes of a wild-type Pasteurella multocida strain and a hfq mutant strain. Comparison of these two transcriptomes allows for determination of differentially expressed genes and therefore those genes controlled by Hfq and sRNAs with which it interacts.
Project description:Objectives: To determine the transcripts that are differentially expressed in a hfq mutant. Hfq is an RNA chaperone that mediates many interactions between regultory RNAs and their mRNA targets. Analysis of the transcriptomes of the Pasteurella multocida wild-type strain and the Pasteurella multocida hfq mutant will allow for identification of genes controlled by hfq and the sRNAs with which it interacts.
Project description:Pasteurella multocida is one of the main pathogens of bovine respiratory disease (BRD), which has brought great losses to the cattle industry. The Pm3 strain was a natural isolates, its pathogenic stronger and sensitive to fluoroquinolones. A highly fluoroquinolone resistant strain Pm64(MIC=64 μg/mL) was formed after continuous induction with subinhibitory concentration (1/2MIC) of enrofloxacin,with the enhanced growth characteristics and large attenuation of pathogenicity in mice. In this study, we report the whole genome sequence and the transcription profile by RNA-Seq of strain Pm3/Pm64. The results showed that there was not too much difference between the two strains at the genome level, 24 and 8 genes could be recognized were added and lost in the gene islands(GIs) of Pm64, Those gene involved in DNA binding, trehalose metabolism, material transportation, capsule synthesis, prophage, amino acid metabolism and other functions. 558 up-regulated and 568 down-regulated genes were found in Pm3 campared with Pm64, from which 20 virulence factor-related DEGs were screened. Genes differentially transcribed were mostly associated with capsular polysaccharide(CPS), lipopolysaccharide (LPS), lipooligosaccharide (LOS), Iron utilization and biofilm composition. We speculated that the main mechanism of virulence attenuation after the formation of resistance of Pm64 comes from the change of the expression profile of these genes. This report helps to elucidate the toxicity targets of P. multocida serogroup A and provide helps for the following research on pathogenesis and antimicrobial drugs development.
Project description:Treatment of bacteria with antibiotics at or close to the inhibitory concentration leads to specific transcriptional responses often affecting target genes and targets pathways. A dataset of transcriptional profiles (compendium) induced by antibiotics with known mode-of-action (MoA) can be used to gain information on the putative MoA of novel substances with unknown MoAs. We used a Pasteurella multocida microarray to generate a compendium of transcriptional profiles and to obtain information on the putative MoA of a novel antibiotic compound. We also show a strong impact of the bacteriostatic antibiotics on P. multocida virulence gene transcription. Keywords: antibiotica treatment, time course Midlog-grown cultures of P. multocida were treated for 10 or 30 min with 8 different antibiotics and one novel compound (thiazin) at minimal inhibitory concentrations (MICs) and were harvested. Control bacteria were not-treated and harvested at approximately the same optical density an OD578 of ~ 0.5. Total RNA was extracted from these samples and labelled with biotin. P. multocida whole genome transcriptional profiling was performed by hybridization on the custom-made Affymetrix microarray according to the manufacturerâs instructions. The experiments were done in triplicates.
Project description:Treatment of bacteria with antibiotics at or close to the inhibitory concentration leads to specific transcriptional responses often affecting target genes and targets pathways. A dataset of transcriptional profiles (compendium) induced by antibiotics with known mode-of-action (MoA) can be used to gain information on the putative MoA of novel substances with unknown MoAs. We used a Pasteurella multocida microarray to generate a compendium of transcriptional profiles and to obtain information on the putative MoA of a novel antibiotic compound. We also show a strong impact of the bacteriostatic antibiotics on P. multocida virulence gene transcription. Keywords: antibiotica treatment, time course
Project description:P. multocida is the causative agent of a wide range of diseases of animals, including fowl cholera in birds. Fowl cholera isolates of P. multocida generally express a capsular polysaccharide composed of hyaluronic acid. There have been reports of spontaneous capsule loss in P. multocida fowl cholera-causing strains but the mechanism by which this occurs has not been determined. In this study, we identified three independent strains that had spontaneously lost the ability to produce capsular polysaccharide. Quantitative RT-PCR showed that these strains had significantly reduced transcription of the capsule biosynthetic genes, but DNA sequence analysis identified no mutations within the cap biosynthetic locus. However, whole genome sequencing of paired capsulated and acapsular strains identified a single nucleotide polymorphism within fis that was present only in the acapsular strain. Sequencing of fis from two independently derived spontaneous acapsular strains showed that each contained a mutation within fis. Complementation of these strains with an intact copy of fis returned normal capsule expression to all strains. Therefore, expression of a functional Fis protein is absolutely required for normal capsule expression in P. multocida.DNA microarray studies comparing one of the acapsular pairs (AL114 to AL1115) identified approximately 30 genes as down-regulated in the mutant; including pfh_B2 which encodes the filamentous hemagglutinin, a known P. multocida virulence factor and the cross protective surface antigen plpE. Biological triplicates of each strain were analysed in a single colour experimental design
Project description:Pasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilised transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida, and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus, and fis and hfq that encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA that encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida.
Project description:The Gram-negative pathogen Pasteurella multocida is responsible for many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by a ribosome binding protein such as ProQ or Hfq. In E. coli and other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to 3’ stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ riboregulation in P. multocida and identify the RNA regions to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-crosslinking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilises, ProQ-dependant sRNAs and transfer RNAs in P. multocida via adenosine enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterised and showed that ProQ bound within the coding sequence of the uncharacterized PmVP161_1121 and within the 3’ region of the sRNA Prrc13.
Project description:P. multocida is the causative agent of a wide range of diseases of animals, including fowl cholera in birds. Fowl cholera isolates of P. multocida generally express a capsular polysaccharide composed of hyaluronic acid. There have been reports of spontaneous capsule loss in P. multocida fowl cholera-causing strains but the mechanism by which this occurs has not been determined. In this study, we identified three independent strains that had spontaneously lost the ability to produce capsular polysaccharide. Quantitative RT-PCR showed that these strains had significantly reduced transcription of the capsule biosynthetic genes, but DNA sequence analysis identified no mutations within the cap biosynthetic locus. However, whole genome sequencing of paired capsulated and acapsular strains identified a single nucleotide polymorphism within fis that was present only in the acapsular strain. Sequencing of fis from two independently derived spontaneous acapsular strains showed that each contained a mutation within fis. Complementation of these strains with an intact copy of fis returned normal capsule expression to all strains. Therefore, expression of a functional Fis protein is absolutely required for normal capsule expression in P. multocida.DNA microarray studies comparing one of the acapsular pairs (AL114 to AL1115) identified approximately 30 genes as down-regulated in the mutant; including pfh_B2 which encodes the filamentous hemagglutinin, a known P. multocida virulence factor and the cross protective surface antigen plpE.