The role of the Cj0440c gene in Campylobacter jejuni
ABSTRACT: Cj0440c encodes a putative transcriptional regulator. To determine the role of Cj0440c in C.jejuni, we knocked out Cj0440c in the wild-type strain (S) to obtain the Cj0440c mutants (SM). Then we compared the transcriptome of the Cj0440c mutant with that of the parent strain using DNA microarray. These comparisons identified 19 genes that showed a≥2-fold change in expression in SM. The differentially expressed genes in SM encode proteins involved in flagellar biosynthesis, O-linked glycosylation and hypothetical proteins with unknown fuctions. Cj0440c may regulate flagellar structural element expression or as a compenent of flagellar complex co-expressed with other flagellar genes. Subsequent experiments demonstrated that inactivation of Cj0440c affected corresponding phenotypes of C.jejuni, including broken flagella, weaker motility and reduced colonization ability in chickens. These findings indicate that Cj0440c governs the expression of multiple genes related to flagellar biosynthesis and O-linked glycosylation. This study provides favorable evidence for completing the information of the Campylobacter jejuni genome. An eight chip study using total RNA recoverd from four separate wild-type cultures of Campylobacter jejuni NCTC111168 (S) and four separate cultures of a mutant strain, Campylobacter jejuni NCTC11168 delta- Cj0440c (SM), in which Cj0440c is deleted. Each chip measures the expression level of 1634 genes from Campylobacter jejuni NCTC11168.
Project description:Campylobacter, a major foodborne pathogen, is increasingly resistant to macrolide antibibotics. Previous findings suggested that development of macrolide resistance in Campylobacter requires a multi-step process, but the molecular mechanisms involved in the process are not known. In our study, erythromycin-resistant C. jejuni mutant (R) was selected in vitro by stepwise exposure of C. jejuni NCTC11168(S) to increasing concentrations of erythromycin.The resistant were subjected to microarray and the the global transcriptional profile was analyzed. In this series, DNA microarray was used to compare the gene expression profiles of the macrolide-resistant strain with its parent wild-type strain NCTC11168. A large number of gene showed significant changes in R. The up-regulated genes in the resistant strains are involved in miscellaneous periplasmic proteins, efflux protein and putative aminotransferase, while the majority of the down-regulated genes are involved in electron transport, lipoprotein, heat shock protein and unknown function proteins. The over-expression of efflux pump and periplasmic protein was involved in the development of resistance to macrolide in C. jejuni. An eight chip study using total RNA recovered from four separate resistant-type cultures of Erythrocin-resistant Campylobacter jejuni NCTC111168 (R) and four separate cultures of Campylobacter jejuni NCTC111168 (S). Each chip measures the expression level of 1634 genes from Campylobacter jejuni NCTC11168.
Project description:Cj0440c, a putative transcriptional regulator, was over-expressed in the high-level erythromycin-resistant (Eryr) Campylobacter jejuni strains. To determine the role of Cj0440c on the development and fitness of erythromycin resistance in C. jejuni, we knocked out Cj0440c in Eryr strain (R) to obtain the Cj0440c mutants (RM). Then we compared the transcriptome of the Cj0440c mutant with that of the parent strain using DNA microarray. These comparisons identified 9 genes that showed a ≥2-fold change in expression in RM. The differentially expressed genes in RM are related to flagellar biosynthesis and unknown functions. What's more, katA, encoding catalase, down-regulated in RM. Cj0440c may progress flagellar genes expression, help to escape drug pressure and disseminate and colonize smoothly, and Cj0440c in Eryr Campylobacter may protect bacteria from harmful oxygen stress from the host immune system, other microorganism in host intestinal and its own products. These findings indicate that Cj0440c is essential for the fitness (growth) of resistant C. jejuni by controlling the expression of several genes involved in flagellar assembly and catalase, enhancing cell motility for colonization and invasion under the pressure of drug. This study widened our understanding on the molecular mechanism of resistance and provides scientific reference for drug research and application. An eight-chip study using total RNA recoverd from four separate resistant-type cultures of Erythrocin-resistant Campylobacter jejuni NCTC 111168 (R) and four separate cultures of a mutant strain, erythrocin-resistant Campylobacter jejuni NCTC 11168 delta- Cj0440c (RM), in which Cj0440c is deleted. Each chip measures the expression level of 1634 genes from Campylobacter jejuni NCTC 11168.
Project description:A highly pathogenic Campylobacter jejuni clone has recently emerged as the major cause of Campylobacter-associated sheep abortion in the U.S. and is also associated with foodborne gastroenteritis in humans. A distinct phenotype of this clone is its ability to induce bacteremia and abortion. To facilitate understanding the pathogenic mechanisms of this hyper virulent clone, the differences in global gene expression patterns between this hyper virulent clone (IA3902) and a non-abortifacient strain (NCTC 11168) were compared by DNA microarray. One-condition experiment, IA3902 vs NCTC11168. Biological replicates: 3 IA3902 , 3 NCTC11168. One replicate per array.
Project description:Expression arrays comparing Campylobacter jejuni NCTC11168 during growth in the cecum of germ-free C57 BL/6 IL-10 knockout mice to C. jejuni NCTC11168 during growth in Bolton broth. Four biological replicates comparing C. jejuni NCTC11168 growth in vivo to in vitro. Two biological replicates were dye swaps.
Project description:During colonization in the host gastrointestinal tract, the enteric bacteria Campylobacter jejuni is exposed to a variety of signaling molecules including the catecholamine hormones epinephrine (Epi) and norepinephrine (NE). NE has been determined to stimulate the growth of C. jejuni as well as increase its pathogenicity. To investigate the mechanisms of NE or Epi on the biology of C. jejuni, the global gene expression profiles of C. jejuni NCTC 11168 cultured in iron-restricted medium were analyzed in response to NE or Epi. Totally, 183 and 156 genes were differentially expressed by NE and Epi respectively, with 102 differentially expressed genes common between the two treatments. These genes are involved in diverse cellular functions including iron uptake systems, motility, virulence, oxidative stress response, nitrosative stress tolerance, enzyme metabolism, DNA repair and metabolism and ribosomal protein biosynthesis. Adherence to and invasion of Caco-2 cells by C. jejuni were enhanced upon exposure to NE or Epi. These results indicated that NE and Epi have similar effects on the gene expression of C. jejuni and that the effects on gene expression may contribute to elucidate the mechanisms on interaction between host and C. jejuni. Transcriptional profiles were analyzed using microarray to compared the epinephrine (Epi) or norepinephrine (NE) treated and untreated Campylobacter jejuni NCTC 11168. NE or Epi treated and untreated cultures of C. jejuni NCTC 11168 were collected at the mid-log phase (～36 h cultures). Three (for NE or Epi treated culture) or four (for untreated control culture) independent biological replicates were performed. Total RNA was extracted using RiboPure™-Bacteria Kit (Ambion, Life Technologies) according to the manufacturer’s instructions. The quality and quantity of total RNA were determined by an Agilent Bioanalyzer 2100. Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color, following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany). Each Slide was hybridized with 600ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA, US) in Hybridization Oven, according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Slides were scanned by Agilent Microarray Scanner with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit. Data were extracted with Feature Extraction software 10.7. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0. The genes with fold change ≥ 1.5 and P < 0.05 were selected as differentially expressed.
Project description:Campylobacter jejuni is a widespread pathogen responsible for most of the food-borne gastrointestinal diseases in Europe. For pathogen control in the food industry, the use of natural antimicrobial molecules is a promising strategy to avoid antibiotic treatments. Isothiocyanates are natural antimicrobial compounds which also display anti-cancer activity. Several studies described the chemoprotective effect of isothiocyanates on eukaryotic cells, but the antimicrobial mechanism is still poorly understood. We investigated the early cellular response of C. jejuni to benzylisothiocyanate (BITC) by both transcriptomic and physiological (respirometry, ATP content measurements and isolations of aggregated proteins). To characterize the transcriptomic early response to benzylisothiocyanate, C. jejuni NCTC11168 were grown in 100 ml flasks containing 25 ml of MEMα medium plus 20 mM sodium pyruvate. At mid-log phase, 2µg/mL benzylisothiocyanate in ethanol, or the same volume of ethanol (control) was added to the flasks for 10 or 15 min prior to total RNA extraction and purification. Samples were then processed for microarray hybridization. Microarray data was acquired from two (10 minutes assay) or three (15 minutes assay) independent biological replicates and 6 to 9 technical replicates for each biological replicate (total number of measurement per gene = 42).
Project description:Campylobacter jejuni is one of the most important causes of food-borne diseases in industrialized countries. It is known that amino acids are important nutrient source for this pathogen, because C. jejuni lacks enzymes related to glycolysis. However, the characteristics on metabolism of C. jejuni grown in the nutrient restricted medium with a specific amino acid is not fully elucidated. This study shows that C. jejuni NCTC11168 grew well in the nutrient restricted medium containing serine, aspartate, glutamate, and proline. The single subtraction of serine significantly reduced the growth, while three other amino acids did not, suggesting the priority of serine among the four amino acids. In the transcriptomic analysis of C. jejuni NCTC11168 grown in medium with serine as a main energy source. Serine seemed to be sensed by some chemoreceptors and the C. jejuni might reached an adaptation stage with active growth. That is, the expression of flagellar assembly components was downregulated and the biosynthesis of multiple amino acids and nucleotide sugars were upregulated. These data suggest the higher requirement of serine as a nutrient of C. jejuni NCTC11168. Overall design: Serine induced gene expression in Campylobacter jejuni was measured at 8 hours after exposure to dose of 20mM.
Project description:The genome-wide Campylobacter jejuni transcriptional response under iron replete and iron limited conditions was characterized using RNA-seq. We have identified 111 novel C. jejuni 5'UTRs and mapped 377 co-transcribed genes into 230 transcriptional operons. In contrast to previous microarray results, the C. jejuni iron stimulon is less extensive than previously believed and consists of 77 iron activated genes and 50 iron repressed genes. As anticipated, the iron repressed genes are primarily those involved in iron acquisition or oxidative stress defense. Interestingly, these experiments have revealed that iron is an important modulator of flagellar biogenesis with almost all the components of the flagella found to be iron activated. Given that motility is a well-known C. jejuni colonization factor, this suggests that there is an important regulatory coupling of flagellar biogenesis and iron level in C. jejuni. In addition we have identified several consensus mutations in the C. jejuni NCTC11168 strain that are widespread in the Campylobacter research community and which may explain conflicting phenotypic reports for this strain. Comparative analysis of iron responsive genes with the known Fur regulon indicates that many iron responsive genes are not Fur responsive; suggesting that additional iron regulatory factors remain to be characterized in C. jejuni. Further analysis of the RNA-seq data identified multiple novel transcripts including 19 potential ncRNAs. The expression of selected ncRNAs was confirmed and quantified with qRT-PCR. The qRT-PCR results indicate that several of these novel transcripts are either Fur and/or iron responsive. The fact that several of these ncRNAs are iron responsive or Fur regulated suggests that they may perform regulatory roles in iron homeostasis.
Project description:This study investigates the CsrA regulon of the food-borne pathogen Campylobacter jejuni. Direct RNA binding targets of CsrA in two strains of C. jejuni, NCTC11168 and 81-176, were determined using RIP-seq. Identification of CsrA binding sites in two C. jejuni strains using RIP-seq