Abstract
RNA thermometers (RNATs) trigger bacterial virulence factor expression in response to the temperature shift on entering a warm-blooded host. At lower temperatures these secondary structures sequester ribosome-binding sites (RBSs) to prevent translation initiation, whereas at elevated temperatures they “melt” allowing translation. Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide yet little is known about how it interacts with the host including host induced gene regulation. Here we demonstrate that an RNAT regulates a C. jejuni gene, Cj1163c or czcD, encoding a member of the Cation Diffusion Facilitator family. The czcD upstream untranslated region contains a predicted stem loop within the mRNA that sequesters the RBS to inhibit translation at temperatures below 37°C. Mutations that disrupt or enhance predicted secondary structure have significant and predictable effects on temperature regulation. We also show that in an RNAT independent manner, CzcD expression is induced by Zn(II). Mutants lacking czcD are hypersensitive to Zn(II) and also over-accumulate Zn(II) relative to wild-type, all consistent with CzcD functioning as a Zn(II) exporter. Importantly, we demonstrate that C. jejuni Zn(II)-tolerance at 32°C, a temperature at which the RNAT limits CzcD production, is increased by RNAT disruption. Finally we show that czcD inactivation attenuates larval killing in a Galleria infection model and that at 32°C disrupting RNAT secondary structure to allow CzcD production can enhance killing. We hypothesise that CzcD regulation by metals and temperature provides a mechanism for C. jejuni to overcome innate immune system-mediated Zn(II) toxicity in warm-blooded animal hosts.
Highlights
The Gram-negative bacterium Campylobacter jejuni is a globally significant cause of gastroenteritis with an estimated annual incidence of 166 million cases [1]
Sensing the elevated warm-blooded host body temperature through structural changes in so-called RNA thermometers is a recognised virulence strategy in bacterial pathogens and allows appropriate production of virulence factors in the host environment. We demonstrated that this regulatory mechanism is used by Campylobacter jejuni, a globally significant cause of human gastroenteritis, to control production of the CzcD protein that we show acts as a Zn(II) exporter providing tolerance to high levels of Zn(II)
Using a simple larval infection model we demonstrated that CzcD enhances larval killing and disrupting the RNA thermometer to allow CzcD production enhanced killing at lower temperatures
Summary
The Gram-negative bacterium Campylobacter jejuni is a globally significant cause of gastroenteritis with an estimated annual incidence of 166 million cases [1]. In developing countries disease is often endemic with symptomatic infections in infants but not in older age groups, presumably due to development of acquired immunity through repeated exposure [2]. In developed countries C. jejuni infections are typically symptomatic, occur sporadically across all age groups and are commonly transmitted via food derived from birds and animals where it is considered to be a commensal. The consumption of poultry, in particular chicken, is a major source of infection [3]. There are rare but serious infection sequelae including the autoimmune mediated peripheral neuropathy Guillain-Barresyndrome [4]. The public health significance of campylobacteriosis underlines the urgent need for new strategies to reduce infections. The mechanisms employed by C. jejuni to adapt to its intraand extracellular lifestyles and to colonise and infect its hosts are relatively poorly understood
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