Long-term survival of Campylobacter jejuni at low temperatures is dependent on polynucleotide phosphorylase activity

Abstract

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10 degrees C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Deltapnp) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42 degrees C). trans-Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10 degrees C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

FIG. 1.

Construction of different vectors used in this study and listed in Table 1: pNH01 (A), pNH02 (B), pNH03 (C), and pNH04 (D).

FIG. 2.

(A) Genetic organization of the pnp CJJ81176_1270 cluster showing the putative transcriptional start site, localization of the insertion of aphA3 within the pnp gene, putative transcriptional terminator site, and position of the different primers used in this study. Kn^R, kanamycin resistance. (B) RT-PCR assays performed using C. jejuni 81-76 mRNA. RT experiments were performed with random hexamer primers, and PCR was done with the primer pairs MO005/MO006 (lane 1), MO007/MO006 (lane 2), MO005/MO008 (lane 3), MO007/MO008 (lane 4), and MO008/MO009 (lane 5). A 1-kb DNA ladder (BioLab) is shown on the left.

FIG. 3.

Survival of C. jejuni strains F38011, 81-76, and their pnp mutant derivatives F38PNP and 81PNP at 42°C (A), 10°C (B), and 4°C (C) under microaerophilic conditions. The values plotted are means ± standard deviations (error bars).

FIG. 4.

Survival of C. jejuni strains F38011, 81-76, and their pnp mutant derivatives F38PNP and 81PNP at 37°C (A), 10°C (B), and 4°C (C) in the ambient atmosphere. The values plotted are means ± standard deviations (error bars).