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 jejuniis 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 aC.jejunigene, Cj1163c orczcD, encoding a member of the Cation Diffusion Facilitator family. TheczcDupstream untranslated region contains a predicted stem loop within the mRNA that sequesters the RBS to inhibit translation at temperatures below 37 degrees 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 lackingczcDare 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 thatC.jejuniZn(II)-tolerance at 32 degrees C, a temperature at which the RNAT limits CzcD production, is increased by RNAT disruption. Finally we show thatczcDinactivation attenuates larval killing in a Galleria infection model and that at 32 degrees C disrupting RNAT secondary structure to allow CzcD production can enhance killing. We hypothesise that CzcD regulation by metals and temperature provides a mechanism forC.jejunito overcome innate immune system-mediated Zn(II) toxicity in warm-blooded animal hosts.
Author summary 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 byCampylobacter 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). From published data, Zn(II) exporters are known to enable bacterial pathogens to overcome host-induced Zn(II) poisoning-a newly discovered innate immune response to infection. Indeed using a simple larval infection model we demonstrated that CzcD enhances larval killing and disrupting the RNA thermometer to allow CzcD production also enhanced killing at lower temperatures. These findings indicate that CzcD temperature regulation enablesC.jejunito overcome host induced Zn(II) toxicity during colonisation of animal hosts including humans. Our results open up a novel area of the host-pathogen interface for investigation in this poorly understood human pathogen. It may also be possible to exploit these findings to develop metal-based intervention strategies to reduce both levels ofCampylobacterin the food chain and consequent zoonotic infections.