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 theC. 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 ahpCencoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences contained flhB and motBhomologs, 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 theahpC 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. TheC. jejuni AhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.

The iron-induced ferredoxin FdxA of Campylobacter jejuni is involved in aerotolerance

“…The ahpC^fdxA intergenic region was PCR-ampli¢ed from C. jejuni strain 81116 using Expand polymerase mix (Roche Diagnostics), checked by sequencing for the absence of incorporation errors, and cloned in both orientations into the BamHI site upstream of the promoterless lacZ gene in pMW10. This resulted in constructs pAV197 (ahpC: :lacZ) [7] and pAV198 (fdxA: : lacZ). As C. jejuni 81116 does not accept plasmid DNA isolated from E. coli [13], reporter plasmids were transformed into C. jejuni strain 480 using electroporation [14].…”

“…ROS have the ability to damage DNA, proteins and lipids, and cells have oxidative stress defense mechanisms to inactivate ROS [2]. C. jejuni contains three proteins active in ROS inactivation: the iron-containing superoxide dismutase SodB [3^5], and the peroxide stress defense proteins catalase (KatA) [6] and alkyl hydroperoxide reductase (AhpC) [7]. The C. jejuni peroxide stress defense genes katA and ahpC are repressed by PerR in response to iron, whereas regulation of SodB expression has not yet been described [7,8].…”

“…C. jejuni contains three proteins active in ROS inactivation: the iron-containing superoxide dismutase SodB [3^5], and the peroxide stress defense proteins catalase (KatA) [6] and alkyl hydroperoxide reductase (AhpC) [7]. The C. jejuni peroxide stress defense genes katA and ahpC are repressed by PerR in response to iron, whereas regulation of SodB expression has not yet been described [7,8]. Both the SodB and the AhpC proteins are involved in the survival of Campylobacter under atmospheric oxygen concentrations.…”

“…A Campylobacter coli sodB mutant displayed markedly decreased survival under aerobic conditions [5], and reduced resistance to oxidative stress induced by freeze-thawing [9]. Similarly, a C. jejuni ahpC mutant also showed reduced survival under aerobic conditions [7].…”

“…Examination of the nucleotide sequence downstream of the ahpC gene [7] revealed the presence of motility-associated genes (C. Matz, A.H.M. van Vliet, J.M.…”