Functional Characterization of the Cation Diffusion Facilitator Family members of Campylobacter jejuni

Abstract

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the world and is therefore an important food borne pathogen, yet its virulence mechanisms are poorly understood. Bacteria require transition metals however, if excess, metals are toxic disrupting cellular processes and host induced metal toxicity is a defence mechanism against infection. Bacteria have evolved metal transport and sequestration systems to overcome this toxicity and maintain cellular homeostasis. Given how little we know about C. jejuni colonization and pathogenesis such metal resistance mechanism are an interesting area for future study. This study identified and characterized two putative metal transporters that belong to Cation Diffusion Facilitator family in C. jejuni NCTC 11168. It is predicted that czcD and cj0948c genes encode proteins (termed as CzcD and Cj0948 here) belong to the Cobalt Zinc and Cadmium resistance family and function as metal exporters across the cell membrane. Insertional mutant strains of czcD and cj0948c were constructed in order to test this hypothesis and their sensitivity to a range of metals determined. Strains lacking CzcD or Cj0948 were sensitive to zinc and iron respectively. Genetically complemented strains were constructed for each of these mutants that restored the metal sensitivity to the wild type levels, confirming the phenotypes. This genetic complementation strategy was further utilized to identify specific CzcD residues required for restoration of zinc tolerance. The structure and function of CzcD was compared with that of Yiip, a homologue from Escherichia coli, and the conserved sequences required for the function of Yiip and the regions specific to the C. jejuni CzcD protein were targeted. The results indicated the differences and similarities in relation to the function of these homologues. These findings provide functional data establishing the respective roles of two key players in the regulation of metal homeostasis in this important human pathogen.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jennifer Cavet (Supervisor) & James Linton (Supervisor)