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Exposure of microaerobic Campylobacter jejuni to diffusible molecules from chicken caecal contents

ABSTRACT: Campylobacter jejuni is the major cause of acute gastroenteritis in the developed world. It is usually acquired through contaminated poultry as C. jejuni causes a silent asymptomatic infection of the chicken. Pathogens face different sources of stress during its transit through the gut. In this study, we describe the ability of C. jejuni to survive nitrosative stress at very low oxygen levels that reflect those in hypoxic gut environments. Specifically, we here explore an innovative model of signal recognition during colonization. We use a diffusion capsule to feed small, diffusible molecules from chicken caecal matter into a microaerobic C. jejuni culture to study the transcriptomic changes mounted as response to chemical signals present in the chicken gut. We find that in early stages of exposure to the caecal contents (10 min) the dual component colonization regulator, dccR, plays an important yet not fully understood role. Although the caecal material contains cyanide derived from plant sources, we find no role for a truncated globin (encoded by ctb), which has previously been implicated in resistance to this haem ligand.

ORGANISM(S): Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 Campylobacter jejuni

PROVIDER: GSE39917 | GEO | 2012/08/07

SECONDARY ACCESSION(S): PRJNA172023

REPOSITORIES: GEO

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Project description:Two batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen's RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For the control slide, Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For the dye-swap slide, Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made to compensate for possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA). The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spotted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc. (ImaGene, version 4.0 and GeneSight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the Student's t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.

Project description:Transcriptional analysis (RNA hybridization) for 11168-G (genome sequenced) vs. 11168-O (original), cultured microaerobically or anaerobically (published as "severely O2-limited). Published in Gaynor et al, J. Bacteriology 186:503-517 The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Computed

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Exposure of microaerobic Campylobacter jejuni to diffusible molecules from chicken caecal contents

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