Campylobacter jejuni benefits from the bile salt deoxycholate under low-oxygen condition in a PldA dependent manner

Figures & data

Figure 1. DOC modified C. jejuni wild-type colony morphology on Campylobacter blood free selective plate.

Colonies of C. jejuni 81116 wild-type, C. jejuni ΔpldA and C. jejuni ΔpldA+pldA were grown on (a) Blood Free Campylobacter Selectivity Agar Base plate, (b) Saponin agar charcoal plate, and (c) Saponin agar charcoal + 0.1% DOC plate. Bacterial morphology was visualized after 16 h of incubation under oxygen limited (5% O₂, 10% CO₂, 75% N₂, 10% H₂) conditions at 42°C.

Figure 2. C. jejuni PldA is needed for optimal growth in the presence of DOC, but the oxygen concentration determines whether DOC improves or inhibits C. jejuni growth.

Growth curves of C. jejuni wild-type, C. jejuni ΔpldA and C. jejuni ΔpldA +pldA were generated in HI with or without 0.1% DOC under (a) microaerobic (10% O₂, 10% CO₂, 70% N₂, 10% H₂) or (a) oxygen limited (0.3% O₂, 10% CO₂, 79.7% N₂, 10% H₂) conditions at 42°C. The optical density was measured at the indicated time points. Experiments were repeated three times in duplicate. Data are represented as mean ± SEM. The displayed P values are calculated by comparing the HI vs HI + DOC treatment of the particular strain.****P < .0001, ***P < .001,*P < .1.

Figure 3. Influence of DOC on the membrane integrity of the C. jejuni wild-type and PldA mutant.

Equal amount of bacteria grown with or without DOC at 10 or 0.3% O₂ were stained with a 1:1 mixture of the nucleic acid staining dyes SYTO-9 (Green) and propidium iodide (red). Slides with the stained bacteria were captured using EVOS fluorescent microscope (Thermo Scientific).

Figure 4. Genes affected by the addition of DOC in C. jejuni wild-type and its isogenic ΔpldA mutant.

Heatmap and hierarchical clustering of the highly regulated genes and reported reference genes are presented. The experiments were repeated three times.

Figure 5. Transcription of genes dependent on the availability of DOC and a functional pldA gene.

The heatmap and hierarchical clustering of the highly regulated genes are presented. The RNA-seq experiments were repeated three times with similar results.

Figure 6. Growth curves of wild-type and pldA mutant with or without DOC and the addition of ferric or ferrous iron.

C. jejuni wild-type and ΔpldA were grown (a) without or (b) with DOC with or without Fe²⁺ or Fe³⁺ at 42°C in HI, under oxygen limited (0.3% O₂, 10% CO₂, 79.7% N₂, 10% H₂) conditions. The optical density (Y- as) at the indicated time points are shown. The experiments were repeated three times in duplicate. Data are represented as mean ± SEM. ****P < .0001, **P < .01, *P < .1.

Figure 7. Survival assay to evaluate the susceptibility of C. jejuni wild-type and C. jejuni ΔpldA to oxidative stress.

Survival assays of C. jejuni wild-type and C. jejuni ΔpldA strains were performed in HI with or without 0.1% DOC exposed to 80 nM H₂O₂ under microaerobic conditions for 30 min. After serial dilutions and incubation, CFUs/ml were counted. Data of three independent experiments with three independent preparations of bacterial samples and presented as mean values ± SEM, ****P < .0001, ns P > 0.1.