An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni

Abstract

Microaerophiles like Campylobacter jejuni must resist oxidative stresses during transmission or infection. Growth of C. jejuni 81116 under iron limitation greatly increased the expression of two polypeptides of 26 and 55 kDa. The identification of these proteins by N-terminal amino acid sequencing showed both to be involved in the defense against oxidative stress. The 55-kDa polypeptide was identical to C. jejuni catalase (KatA), whereas the N terminus of the 26-kDa polypeptide was homologous to a 26-kDa Helicobacter pylori protein. The gene encoding the C. jejuni 26-kDa protein was cloned, and the encoded protein showed significant homology to the small subunit of alkyl hydroperoxide reductase (AhpC). The upstream region of ahpC encoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences contained flhB and motB homologs, which are involved in flagellar motility. There was no evidence for an adjacent homolog of ahpF, encoding the large subunit of alkyl hydroperoxide reductase. Reporter gene studies showed that iron regulation of ahpC and katA is achieved at the transcriptional level. Insertional mutagenesis of the ahpC gene resulted in an increased sensitivity to oxidative stresses caused by cumene hydroperoxide and exposure to atmospheric oxygen, while resistance to hydrogen peroxide was not affected. The C. jejuni AhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.

FIG. 1

Effects of iron restriction on total protein profiles of C. jejuni 81116 and AV32 (ahpC mutant) strains. Lane 1, C. jejuni 81116 grown in MEMα (iron restricted); lane 2, C. jejuni 81116 grown in MEMα supplemented with 40 μM Fe(III)SO₄ (iron replete); lane 3, C. jejuni AV32 grown in MEMα (iron limited); lane 4, C. jejuni AV32 grown in MEMα supplemented with 40 μM Fe(III)SO₄ (iron replete). Molecular mass markers (in kilodaltons) are shown on the left. Arrows on the right indicate the 55-kDa KatA and 26-kDa AhpC polypeptides.

FIG. 2

Alignment of C. jejuni AhpC protein (Cj) with AhpC proteins of H. pylori (Hp), Legionella pneumophila (Lp), and E. coli (Ec). Asterisks denote identical residues, and dots indicate conservative substitutions. Regions used for the design of degenerate primers are boxed, and the directions of the resulting primers are indicated by arrows.

FIG. 3

Restriction and gene map of the C. jejuni genomic region containing the ahpC gene. Only a part of the motB gene is shown. The place and direction of insertion of the chloramphenicol resistance gene (Cm^r) used to create the C. jejuni ahpC mutant are also indicated.

FIG. 4

Analysis of the promoter region of the C. jejuni ahpC gene. The mapping of the transcriptional start site of ahpC is shown (left panel). Lane 1, primer extension of C. jejuni mRNA; lanes 2 to 5, sequencing reaction of pMLB4.2 (in the order C, T, A, and G). The right-hand column shows the complementary template sequence, and the transcriptional start site is indicated by an asterisk. The sequence of the ahpC promoter region is shown in the upper panel. The start codons of ahpC and the divergent fdxA gene are indicated, as well as the transcriptional start site (+1) of ahpC. Sequences closely resembling the C. jejuni ς⁷⁰ consensus sequence (Cons) are indicated.

FIG. 4

Analysis of the promoter region of the C. jejuni ahpC gene. The mapping of the transcriptional start site of ahpC is shown (left panel). Lane 1, primer extension of C. jejuni mRNA; lanes 2 to 5, sequencing reaction of pMLB4.2 (in the order C, T, A, and G). The right-hand column shows the complementary template sequence, and the transcriptional start site is indicated by an asterisk. The sequence of the ahpC promoter region is shown in the upper panel. The start codons of ahpC and the divergent fdxA gene are indicated, as well as the transcriptional start site (+1) of ahpC. Sequences closely resembling the C. jejuni ς⁷⁰ consensus sequence (Cons) are indicated.

FIG. 5

β-Galactosidase assays of C. jejuni strain transformants grown under iron-restricted and iron-replete conditions, confirming that iron regulation of ahpC and katA expression is achieved at the transcriptional level. Error bars represent standard deviations.

FIG. 6

Percentage survival of C. jejuni 81116 wild-type and ahpC mutant stationary-phase cells kept under normal atmospheric oxygen conditions at 37°C. The graph shows results of a single representative experiment typical of the six that were carried out.