The RNase R from Campylobacter jejuni has unique features and is involved in the first steps of infection

Abstract

Bacterial pathogens must adapt/respond rapidly to changing environmental conditions. Ribonucleases (RNases) can be crucial factors contributing to the fast adaptation of RNA levels to different environmental demands. It has been demonstrated that the exoribonuclease polynucleotide phosphorylase (PNPase) facilitates survival of Campylobacter jejuni in low temperatures and favors swimming, chick colonization, and cell adhesion/invasion. However, little is known about the mechanism of action of other ribonucleases in this microorganism. Members of the RNB family of enzymes have been shown to be involved in virulence of several pathogens. We have searched C. jejuni genome for homologues and found one candidate that displayed properties more similar to RNase R (Cj-RNR). We show here that Cj-RNR is important for the first steps of infection, the adhesion and invasion of C. jejuni to eukaryotic cells. Moreover, Cj-RNR proved to be active in a wide range of conditions. The results obtained lead us to conclude that Cj-RNR has an important role in the biology of this foodborne pathogen.

Keywords: Adhesion; Campylobacter; Foodborne Pathogen; Invasion; RNA Catalysis; RNA Metabolism; Ribonuclease.

FIGURE 1.

A, schematic representation of the domain organization of RNase R proteins from E. coli and C. jejuni. N, N terminus; C, C terminus; HTH, helix-turn-helix. B, phylogenetic tree of RNase R from E. coli, S. enterica, C. jejuni, S. aureus, S. pneumoniae, M. genitalium, B. subtilis, and H. pylori.

FIGURE 2.

Salt dependence of Cj-RNR. A, 1 nm recombinant protein was incubated with 10 nm poly(A) at 37 °C for 5 min in a reaction buffer with different salt concentrations as indicated. Samples were taken during the reaction at the time points indicated. B, determination of the activity of Cj-RNR. Error bars indicate mean ± S.D.

FIGURE 3.

pH dependence of Cj-RNR. A, 1 nm recombinant protein were incubated with 10 nm poly(A) at 37 °C for 5 min in a reaction buffer with different pH, ranging from 5.4 to 9. Samples were taken during the reaction at the time points indicated. Ctrl, control. B, determination of the activity of Cj-RNR. Error bars indicate mean ± S.D.

FIGURE 4.

Divalent metal ion dependence of Cj-RNR. A, 25 nm recombinant protein were incubated with 10 nm poly(A) at 37 °C for 10 min in a reaction buffer with different divalent metal ions, which are indicated in the figure. Ctrl, control. B and C, 1 nm recombinant protein was incubated with 10 nm poly(A) at 37 °C for 5 min in a reaction buffer with different Mg²⁺ (B) and Mn²⁺ (C) concentrations. Samples were taken during the reaction at the time points indicated. D, determination of the activity of Cj-RNR. Error bars indicate mean ± S.D.

FIGURE 5.

Exoribonucleolytic activity of Cj-RNR at different temperatures. 25 nm recombinant protein was incubated with 10 nm poly(A) at 4, 30, 37, and 42 °C for 10 min. Samples were taken during the reaction at the time points indicated. Ctrl, control.

FIGURE 6.

Exoribonucleolytic activity of Cj-RNR using ssRNA (A) or dsRNA (B) molecules. The recombinant protein was incubated with 10 nm RNA substrates at 37 °C for 20 min. Samples were taken during the reaction at the time points indicated. The protein concentration used is indicated in the figure. Ctrl, control.

FIGURE 7.

Determination of the activity of Cj-RNR at different temperatures. The activity of the protein was determined at 4, 30, 37, and 42 °C as described under “Experimental Procedures” using three different synthetic substrates: poly(A), 16ss, and the double-stranded 16–30ds. All the activity assays were performed in triplicate. Error bars indicate mean ± S.D.

FIGURE 8.

Exoribonucleolytic activity of Cj-RNR with a DNA molecule showing comparison with E. coli RNase II (Ec RNB) and RNase R (Ec RNR). 10 nm of each protein was incubated with 30 nm DNA at 37 °C for 60 min. Samples were taken during the reaction at the time points indicated. Ctrl, control.

FIGURE 9.

Exoribonucleolytic activity of Cj-RNR with tRNA^Ser and rRNA molecules. 50 nm of the recombinant protein was incubated with 10 nm tRNA^Ser and rRNA at 37 °C for 30 min. Samples were taken during the reaction at the time points indicated. Ctrl, control.

FIGURE 10.

Growth (left panel) and viability (right panel) of C. jejuni 81–176 wild-type strain (in blue) and Δrnr mutant strain (in red) at optimal temperature of growth (37 °C) (A) and at minimal temperature of growth (32 °C) (B). The data represent the mean of the experiments under each condition, with standard deviations shown. OD, optical density.

FIGURE 11.

Adhesion and invasion ability of C. jejuni 81–176 wild-type and Δrnr strains. The results are expressed as the ratio between wild-type and Δrnr strains. Error bars indicate mean ± S.D.