Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni

Abstract

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.

Keywords: 2D-electrophoresis; Campylobacter jejuni; chick colonization; in vitro virulence tests; polynucleotide phosphorylase.

FIGURE 1

Relative expression levels of C. jejuni 81-176 pnp gene at different conditions. Gene expression has been estimated using RT-qPCR and the comparative critical threshold (ΔΔC_T) method. The rpoA gene was used as the internal control, and the expression at 37°C in mid-log phase as the calibrator. Error bars represent the standard deviations from the mean of three independent experiments.

FIGURE 2

Mean generation time of the parental strain (solid circles) versus the mutant strain (open circles). C. jejuni was cultivated in BHI broth and incubated in microaerobic condition at 32, 34, 37, and 42°C. Experiments were performed three times independently.

FIGURE 3

Motility phenotypes of C. jejuni strains. Swimming ability was assessed on BHI agar containing 0.4% agar. Strains were inoculated into BHI motility agar and incubated for 48 h at 37°C under microaerobic conditions. (A) Mean diameters of four independent experiments were represented. (B) Motility agar obtained after an incubation at 37°C during 48 h. (C) Motility assay of the parental C. jejuni F38011 strain (1), the F38Δpnp complemented strain (2), and the mutant strain F38Δpnp (3), on BHI plates containing 0.4% agar.

FIGURE 4

Adhesion and invasion of C. jejuni in Ht-29 cells. (A) Effect of mutation of the pnp locus on C. jejuni adhesion to Ht-29 human intestinal epithelial cells. (B) Ht-29 cell invasion assay. Cells were infected with the parental and mutant strains for 3 h, and invasion values were calculated from the number of bacteria that survived 2 h of incubation in the presence of gentamicin. The experiment was performed in duplicate from at least three independent cultures. The values plotted represent means plusmn; ± standard deviations (error bars). Statistical significance was assessed with an unpaired Student’s t-test. ***P < 0.02.

FIGURE 5

Effect of the PNPase mutation on chick gut colonization by C. jejuni. The experiment included three groups of birds. Two days old chicks were orally gavaged with 1.4 to 1.5 × 10⁷ CFU of C. jejuni 81-176 (red squares) or 81-176 derivative strain lacking production of PNPase (blue squares). Colonization levels were measured by enumeration of bacteria present in ceca on days 7, 14, 21, and 28 post-inoculation (LOG₁₀ CFU/g of ceca). Each dot represents the load of C. jejuni in the cecum of an individual chick. The geometric mean of the bacterial loads from each set of chicks is denoted by the horizontal bar. The line designates the detection limit of 2 × 10² CFU/g of ceca. Statistical analysis was performed using the Mann–Whitney test (mean and P-value < 0.01). *Significant difference between the parental and pnp mutant strains.

FIGURE 6

(A) Two-dimensional electrophoresis gels of acidic proteins extracted from C. jejuni strain 81-176 (a) and the pnp mutant (b) derived from this strain. Proteins were extracted from bacteria cultivated in BHI at 42°C collected when OD600 reached 0.2. (B) Two-dimensional protein gel analysis of basic proteins extracted from C. jejuni strain 81-176 (c) and the pnp mutant (d) derived from this strain. Proteins were extracted from bacteria cultivated in BHI at 42°C collected when OD₆₀₀ reached 0.2.

FIGURE 7

Comparison of relative transcript levels of luxS, peb3, katA, and hsp90 genes between C. jejuni pnp mutant and the parental strain, assessed by quantitative RT-PCR. Each bar represents relative expression level of a gene in the mutant strain according to the 2AACj method described by Livak and Schmittgen (2001). The internal reference gene used was rpoA and the calibrator condition was the expression in the wild-type strain (equivalent to 1). Each value is the mean of at least three independent RNA extractions and error bars indicate the standard deviations.

FIGURE 8

Poly(A) degrading activity in C. jejuni cell lysates. Degradation of [³H]poly(A) was quantified by scintillation counting [³H]nucleotide released into acid soluble form. Dependence of the reaction on lysates of the parental strain (circle) and the pnp mutant (square) was measured in the presence (closed) and absence (open) of 10 mM KPO₄ to distinguish hydrolytic from phosphorolytic activities.