Characterisation of a virulence associated metal resistance operon in the food-borne pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is the primary bacterial cause of foodborne gastroenteritis globally. Within the food chain and during colonisation and infection of its hosts, C. jejuni must tolerate a variety of environmental stresses, both inside and outside the host, including fluxes in metal levels and exposure to reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive chlorine species (RCS) associated with host innate immune defences and food processing. Within the C. jejuni NCTC 11168 genome, czcD and copA are located within a four gene operon, cj1164c-cj 1161c (designated cmr), encoding zinc and copper exporters from the Cation Diffusion Facilitator (CDF) and P1B-type ATPase families, respectively. In addition to zinc and copper resistance, this work has demonstrated that both CzcD and CopA in C. jejuni also contribute to tolerance to ROS and bile deoxycholate, whilst CopA was also provides resistance to RNS. The regulation of the cmr operon promoter (Pcmr) was investigated, with zinc, copper and cobalt shown to induce transcription as well as ROS, consistent with the operon being associated with resistance to these stresses. Notably, a role for Fur in regulating expression from Pcmr in response to zinc and ROS has been demonstrated, whilst PerR has also been shown to influence expression in response to ROS. Accumulation of manganese is reported to be important in ROS resistance in bacteria and these studies have demonstrated that manganese accumulation is important for protecting C. jejuni from oxidative stress. However, despite zinc exposure causing manganese-depletion in C. jejuni, manganese does not provide protection against zinc-toxicity. As part of these investigations, two systems involved in C. jejuni NCTC 11168 manganese import have been identified, with both cj0141c that encodes the deduced inner membrane permease of an MntB-like ABC transporter and zupT (cj0263) that encodes a ZIP family transporter being required for maintaining cellular manganese levels, with cj0141c being essential for growth in the absence of manganese or zinc supplementation. This report confirmed a requirement for Cj0141c for manganese accumulation in C. jejuni to levels that are sufficient to confer protection against oxidative stress and aerobic conditions. Overall, this study has further defined the roles of CzcD and CopA in protecting C. jejuni from antimicrobial stresses and the factors associated with regulating their production, in addition to identifying the systems associated with maintaining manganese homeostasis in this important foodborne pathogen.