Transcriptome analysis of aerotolerant and aerosensitive Campylobacter jejuni strains in aerobic conditions

Abstract

Aerotolerance is vital for the survival of Campylobacter jejuni in the food supply, but the genetic mechanisms underlying aerotolerance remain unclear. This study compares differential gene expression in one aerotolerant and one aerosensitive strain of C. jejuni (WP2202 and T1-21 respectively) in aerobic vs. microaerobic conditions using RNA-Seq technology. The results show that the aerotolerant strain differentially regulated a greater number of genes under aerobic vs. microaerobic conditions as compared to the aerosensitive strain, particularly during the first 6 h of exposure. Differential analysis between aerobic and microaerobic conditions showed that COG category S (genes with unknown functions) had the highest number of DEGs across all timepoints in both strains. When category S was excluded, COG category J (translation, ribosomal structure, and biogenesis) had the highest number of DEGs between aerobic vs. microaerobic conditions with downregulated genes occurring at most timepoints in the two strains. Several previously characterized oxidative stress genes were differentially regulated in both strains in response to aerobic conditions. Both strains upregulated multiple heat shock genes in response to oxygen exposure, supporting the hypothesis that these genes might play a role in the oxidative stress response. A few genes involved in iron acquisition or transport were significantly upregulated under aerobic conditions in the aerosensitive strain, potentially forming reactive oxygen radicals due to increased iron levels. A spike in gene expression after 12 h of oxygen exposure was noted for both strains in various genes across the genome. This study demonstrates differences in differential gene expression between an aerotolerant and an aerosensitive strain in response to exposure to atmospheric oxygen and sheds light into understanding C. jejuni aerotolerance. Numerous genes with potential roles in C. jejuni aerotolerance were identified which provides new avenues for future research. In particular, the benefits and drawbacks of iron to the oxidative stress response and the links between the oxidative stress response and the expression of heat shock genes require further investigation.

Keywords: Campylobacter; aerotolerance; differential gene expression; oxidative stress; transcriptome.

Figure 1

Comparison of gene regulation in the aerosensitive C. jejuni strain T1-21 and the aerotolerant strain WP2202, excluding plasmid genes. (A) The percentage of genes regulated at each timepoint. (B) The number of genes significantly regulated at all timepoints. Significance was defined as FDR p-values less than 0.05 and fold-change magnitudes of 1.5 or greater.

Figure 2

Number of genes significantly up- or down-regulated in different COG categories. Plasmid genes are excluded. Datapoints represent gene regulation in the aerosensitive C. jejuni T1-21 and aerotolerant WP2202 as a function of time. COG Category S was removed to allow for more effective scaling of y-axes. Significance was defined as FDR p-values less than 0.05 and fold-change magnitudes of 1.5 or greater. (A) RNA processing and modification. (B) Chromatin structure and dynamics. (C) Energy production and conversion. (D) Cell cycle control, cell division, and chromosome partitioning. (E) Amino acid transport and metabolism. (F) Nucleotide transport and metabolism. (G) Carbohydrate transport and metabolism. (H) Coenzyme transport and metabolism. (I) Lipid transport and metabolism. (J) Translation, ribosomal structure, and biogenesis. (K) Transcription. (L) Replication, recombination, and repair. (M) Cell wall/membrane/envelope biogenesis. (N) Cell motility. (O) Posttranslational modification, protein turnover, chaperones. (P) Inorganic ion transport and metabolism. (Q) Secondary metabolites biosynthesis, transport and catabolism. (R) General function prediction only. (T) Signal transduction mechanisms. (U) Intracellular trafficking, secretion, and vesicular transport. (V) Defense mechanisms. (W) Extracellular structures. (X) Mobilome: prophages, transposons. (Y) Nuclear structure. (Z) Cytoskeleton.

Figure 3

Fold changes in ahpC, sodB, and katA expression in in the aerosensitive C. jejuni strain T1-21 and the aerotolerant strain WP2202. “NS” indicates nonsignificant data. Significance is defined as an FDR p-value less than 0.05. The y-axes are scaled differently on each graph due to variability in fold-change magnitudes.

Figure 4

Heatmap of fold changes at each timepoint for genes studied previously in the literature for C. jejuni aerosensitive strain T1-21 and aerotolerant strain WP2202. Genes were selected based on prior studies suggesting their involvement in aerotolerance and/or the oxidative stress response. Red indicates downregulation and green indicates upregulation. Nonsignificant timepoints are included. Average of fragment fold changes were taken for each timepoint of the WP2202 flaA fragments (flaA_1, flaA_2, flaA_3, and flaA_4).

Figure 5

Fold changes in the heat shock response genes clpB, dnaK, groL, groS, grpE, and hrcA in the aerosensitive C. jejuni strain T1-21 and the aerotolerant strain WP2202. “NS” indicates nonsignificant data. Significance is defined as an FDR p-value less than 0.05.

Figure 6

Fold changes in the expression of iron regulatory genes tpd, hugZ, cirA2, hmuU, and dps in the aerosensitive C. jejuni strain T1-21 and the aerotolerant strain WP2202. “NS” indicates nonsignificant data. Significance is defined as an FDR p-value less than 0.05. The y-axes are scaled differently to account for differences in fold-change magnitude.

Figure 7

Heatmap of fold changes in expression at each timepoint for 18 ribosomal genes in the aerosensitive C. jejuni strain T1-21 and the aerotolerant strain WP2202. Darker red represents a greater magnitude of downregulation. Nonsignificant timepoints are included.

Figure 8

Fold changes of genes that were up- or down-regulated in the C. jejuni aerosensitive strain T1-21 but not the aerotolerant strain WP2202. “NS” indicates nonsignificant data. Significance was defined as an FDR p-value less than 0.05. The y-axes were scaled differently on some of the graphs due to differences in fold-change magnitudes.

Figure 9

Confirmation of RNA-Seq results by qPCR. The five scatterplots show Pearson correlation plots for clpB, dnaK, groL, groS, and hmuU in the aerotolerant strain WP2202.