Characterisation of Campylobacter jejuni Proteins Involved in Metal Homeostasis

Abstract

The Gram-negative bacterium Campylobacter jejuni is a globally important cause of food-borne gastroenteritis; however, relative to other similar pathogens such as Escherichia coli or Salmonella, we understand little about how it causes disease. Transition metals play an important role in multiple biological processes and their acquisition is critical for host colonisation and virulence. However, in excess, metals are toxic, leading to disruption of metabolic functions, and hence, maintaining metal homeostasis is essential. In common with other bacterial pathogens, C. jejuni has evolved transport and storage-based solutions to overcome toxicity. This work described herein is focused on two distinct C. jejuni gene loci (cj0948c and cj1164c-cj1161c, the latter known as the cmr operon), both encoding metal exporters (Cj0948c, Cj1163c and Cj1161c) belonging to the Cation Diffusion Facilitator (CDF) or P1B type ATPase families. The Cj0948c CDF family member was confirmed to be required for iron tolerance (and hence named Cie for Campylobacter iron export) and demonstrated to provide protection against reactive oxygen species as well as promoting intra-macrophage survival and virulence in the Galleria mellonella larval infection model. Finally, a detailed site-directed mutagenesis study identified critical residues important for Cie function. For the cj1164c-cj1161c locus, mutational analysis confirmed Cj1162c (CopZ) and Cj1161c (CopA) as copper tolerance determinants and Cj1163c (CzcD) as a zinc tolerance determinant. CzcD and CopA were also shown to promote G. mellonella larval killing, the former in a temperature-dependent manner consistent with the previous data on RNA thermometer-mediated regulation of CzcD production. The regulation of the cmr operon was investigated with important data on the role of the Fur regulator in mediating iron-dependent repression of CzcD production and the potential role of a small RNA produced antisense to the cj1164c transcript also regulating CzcD. Overall, the data further define the function, role in virulence and regulatory inputs into genes involved in maintaining zinc, copper and iron homeostasis in this important bacterial pathogen.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jennifer Cavet (Supervisor) & James Linton (Supervisor)