Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion

Abstract

The putative global posttranscriptional regulator csrA was mutated in Campylobacter jejuni 81-176. The csrA mutant was attenuated in surviving oxidative stress. CsrA also contributed to biofilm formation and adherence to and invasion of INT407 intestinal epithelial cells, suggesting a regulatory role for CsrA in C. jejuni pathogenesis.

FIG. 1.

CsrA is required for full motility. Swarming ability was assessed on MH agar containing 0.4% agar. Strains were inoculated into MH motility agar and incubated for 24 h (A) and 48 h (B) at 37°C under microaerobic conditions. (C) Growth of the 81-176 and 81-176 csrA strains was observed in MH broth and measured by determining the OD₆₀₀. The assay was carried out in triplicate, and one representative of three experiments is shown (**, P ≤ 0.005) with error bars.

FIG. 2.

Deletion of CsrA results in reduced resistance to sources of oxidative stress. The 81-176 (▪), 81-176 csrA (•), and 81-176 csrA/pJF11 (⧫) strains were subjected to oxidative stress by exposure to atmospheric oxygen, whereby the strains were inoculated in flasks at an OD₆₀₀ of ∼0.1 and a 6:1 surface-to-volume ratio and then incubated at 37°C and 100 rpm in an air incubator (A), and various concentrations of hydrogen peroxide in filter discs on MH agar plates (B). One representative, in triplicate, of three experiments is shown (*, P ≤ 0.05; **, P ≤ 0.005) with error bars.

FIG. 3.

CV staining of C. jejuni biofilms. CV-stained biofilms were solubilized in 80% dimethyl sulfoxide (A) and quantitated by determining the OD₅₇₀ (B). One representative, in triplicate, of three experiments is shown (**, P ≤ 0.005) with error bars.

FIG. 4.

Adherence and invasion of INT407 cells. The capacities of the 81-176, 81-176 csrA, and 81-176 csrA/pJF11 strains to adhere to and invade INT407 cells at a multiplicity of infection of 15 were examined in vitro. (A) Adherence is expressed as the percentage of bacteria which had either adhered to or invaded cultured intestinal epithelial cells after a 3-h incubation, compared to the inoculum. (B) Invasiveness is expressed as the percentage of intracellular bacteria surviving gentamicin treatment of the INT407 cells after an additional 2-h incubation, compared to the number of adherent bacteria (to account for differences in adherence among strains). One representative, in triplicate, of three experiments is shown (*, P ≤ 0.05) with error bars.