Characterisation of Cj1164c, a Novel Zinc-finger Protein in the Human Pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide, affecting around 0.5 billion people annually, yet its virulence mechanisms are poorly understood. This bacterium colonises and persists in farm animals, particularly chickens, which are the major source of human campylobacteriosis. Zinc is known to play a central role in the outcome of pathogen-host interactions and bacterial zinc homeostasis proteins represent important virulence factors. Previous studies identified a C. jejuni zinc and copper resistance operon (cj1164c-cj1161c), encoding a P1B-type ATPase associated with copper resistance (Cj1161c), a CopZ metallochaperone-like protein (Cj1162c), a CDF family zinc exporter (Cj1163c or CzcD) recently associated with virulence in a mammalian infection model, and a predicted zinc-finger protein of unknown function (Cj1164c). Cj1164c lacks similarity to previously characterised proteins, but the location of cj1164c within the Campylobacter metal-resistance (cmr) operon indicates a potential role in C. jejuni metal handling. This study focused on the functional characterisation of Cj1164c. C. jejuni mutants with a disrupted cj1164c gene, alone or in combination with czcD, and mutants overexpressing Cj1164c were analysed with respect to metal tolerance, metal accumulation and changes in cmr operon expression. These studies provide further support for the role of CzcD as a zinc exporter that confers zinc resistance and reveal some interplay between zinc export and iron homeostasis. CzcD expression was shown to increase in response to zinc (and cobalt) whilst expression was reduced in response to iron, and loss of czcD caused enhanced growth in the presence of iron at concentrations that are non-inhibitory to wild-type C. jejuni. However, no phenotype concerning metal tolerance or metal accumulation was detected in these studies following the disruption or overexpression of cj1164c. Notably, however, whilst cj1164c was shown to not be required for metal responsive expression from the cmr operon, reporter gene analyses indicated that cj1164c does have some effect on the magnitude of zinc responsiveness of CzcD expression and may act to ‘fine-tune’ operon expression in response to an environmental stimulus other than zinc. For further characterisation, Cj1164c was successfully overexpressed in Escherichia coli and purified using endogenous properties (not-tagged). Cj1164c was co-purified with a stably bound zinc atom associated with the amino-terminus C4-type zinc-finger motif, and a mutant protein lacking the zinc-finger (due to substitution of the cysteines with serines) was purified lacking zinc. Both SEC and SEC-MALS analyses of Cj1164c revealed Cj1164c to mainly exist as a dimer, with a predicted MW of around 20.83 kD. In addition to the C4-type zinc-finger, Cj1164c was shown to possess a second metal-binding site associated with carboxyl-terminus histidines that can coordinate zinc, cobalt, iron and nickel (~1 metal atom per dimer). Moreover, the carboxyl-terminus histidines were shown, by AUC, to be required for protein dimerisation in the absence of zinc and substitution of the histidines to alanines gave greater resistance to protein aggregation in the presence of zinc. Further evidence of Cj1164c metal-binding was also provided by the detection of metal-induced conformational changes by nuclear magnetic resonance spectroscopy. To allow further characterisation of Cj1164c and CzcD in the future, polyclonal antibodies against these proteins were also generated in this study. Understanding the mechanisms by which C. jejuni avoids metal stress offers routes for effective intervention in this important disease.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jennifer Cavet (Supervisor) & James Linton (Supervisor)