Functional characterization of a TerC family protein of
Riemerella anatipestifer
in manganese detoxification and virulence
Authors: Chen Qinyuan , Guo Fang , Huang Li , Wang Mengying , Shi Chunfeng , Zhang Shutong , Yao Yizhou , Wang Mingshu , Zhu Dekang , Jia Renyong , Chen Shun , Zhao Xinxin , Yang Qiao , Wu Ying , Zhang Shaqiu , Tian Bin , Huang Juan , Ou Xumin , Gao Qun , Sun Di , Zhang Ling , Yu Yanling , He Yu , Wu Zhen , Götz Friedrich , Cheng Anchun , Liu Mafeng 
Published: December 2023

ABSTRACT

Manganese (Mn) is an essential element for bacteria, but the overload of manganese is toxic. In a previous study, we showed that the cation diffusion facilitator protein MetA and the resistance-nodulation-division efflux pump MetB are responsible for Mn efflux in the bacterial pathogen
Riemerella anatipestifer
CH-1. However, whether this bacterium encodes additional manganese efflux proteins is unclear. In this study, we show that
R. anatipestifer
CH-1 encodes a tellurium resistance C (TerC) family protein with low similarity to other characterized TerC family proteins. Compared to the wild type (WT), the
terC
mutant of
R. anatipestifer
CH-1 (
∆terC
) is sensitive to Mn(II) intoxication. The ability of TerC to export manganese is higher than that of MetB but lower than that of MetA. Consistently,
terC
deletion (
∆terC
) led to intracellular accumulation of Mn
2+
under excess manganese conditions. Further study showed that
∆terC
was more sensitive than the WT to the oxidant hypoclorite but not to hydrogen peroxide. Mutagenesis studies showed that the mutant at amino acid sites of Glu116 (E116), Asp122 (D122), Glu245 (E245) Asp248 (D248), and Asp254 (D254) may be involved in the ability of TerC to export manganese. The transcription of
terC
was upregulated under excess manganese and downregulated under iron-limited conditions. However, this was not dependent on the manganese metabolism regulator MetR. In contrast to a strain lacking the manganese efflux pump MetA or MetB, the
terC
mutant is attenuated in virulence in a duckling model of infection due to increased sensitivity to duck serum. Finally, comparative analysis showed that homologs of TerC are distributed across the bacterial kingdom, suggesting that TerC exerts a conserved manganese efflux function.

IMPORTANCE

Riemerella anatipestifer
is a notorious bacterial pathogen of ducks and other birds. In
R. anatipestifer
, the genes involved in manganese efflux have not been completely identified, although MetA and MetB have been identified as two manganese exporters. Additionally, the function of TerC family proteins in manganese efflux is controversial. Here, we demonstrated that a TerC family protein helps prevent Mn(II) intoxication in
R. anatipestifer
and that the ability of TerC to export manganese is intermediate compared to that of MetA and MetB. Sequence analysis and mutagenesis studies showed that the conserved key amino sites of TerC are Glu116, Asp122, Glu245, Asp248, and Asp254. The transcription of terC was regulated by manganese excess and iron limitation. Finally, we show that TerC plays a role in the virulence of
R. anatipestifer
due to the increased sensitivity to duck serum, rather than the increased sensitivity to manganese. Taken together, these results expand our understanding of manganese efflux and the pathogenic mechanisms of
R. anatipestifer
.