Manganese Efflux Achieved by MetA and MetB Affects Oxidative Stress Resistance and Iron Homeostasis in Riemerella anatipestifer.

In bacteria, manganese homeostasis is controlled by import, regulation, and efflux. Here, we identified 2 Mn exporters, MetA and MetB (manganese efflux transporters A and B), in Riemerella anatipestifer CH-1, encoding a putative cation diffusion facilitator (CDF) protein and putative resistance-nodulation-division (RND) efflux pump, respectively. Compared with the wild type (WT), ΔmetA, ΔmetB, and ΔmetAΔmetB exhibited sensitivity to manganese, since they accumulated more intracellular Mn2+ than the WT under excess manganese conditions, while the amount of iron in the mutants was decreased. Moreover, ΔmetA, ΔmetB, and ΔmetAΔmetB were more sensitive to the oxidant NaOCl than the WT. Further study showed that supplementation with iron sources could alleviate manganese toxicity and that excess manganese inhibited bacterial cell division. RNA-Seq showed that manganese stress resulted in the perturbation of iron metabolism genes, further demonstrating that manganese efflux is critical for iron homeostasis. metA transcription was upregulated under excess manganese but was not activated by MetR, a DtxR family protein, although MetR was also involved in manganese detoxification, while metB transcription was downregulated under iron depletion conditions and in fur mutants. Finally, homologues of MetA and MetB were found to be mainly distributed in members of Flavobacteriaceae. Specifically, MetB represents a novel manganese exporter in Gram-negative bacteria. IMPORTANCE Manganese is required for the function of many proteins in bacteria, but in excess, manganese can mediate toxicity. Therefore, the intracellular levels of manganese must be tightly controlled. Manganese efflux transporters have been characterized in some other bacteria; however, their homologues could not be found in the genome of Riemerella anatipestifer through sequence comparison. This indicated that other types of manganese efflux transporters likely exist. In this study, we characterized 2 transporters, MetA and MetB, that mediate manganese efflux in R. anatipestifer in response to manganese overload. MetA encodes a putative cation diffusion facilitator (CDF) protein, which has been characterized as a manganese transporter in other bacteria, while this is the first observation of a putative resistance-nodulation-division (RND) transporter contributing to manganese export in Gram-negative bacteria. In addition, the mechanism of manganese toxicity was studied by observing morphological changes and by transcriptome sequencing. Taken together, these results are important for expanding our understanding of manganese transporters and revealing the mechanism of manganese toxicity.

Roles of B739_1343 in iron acquisition and pathogenesis in Riemerella anatipestifer CH-1 and evaluation of the RA-CH-1ΔB739_1343 mutant as an attenuated vaccine.

Iron is one of the most important elements for bacterial survival and pathogenicity. The iron uptake mechanism of Riemerella anatipestifer (R. anatipestifer, RA), a major pathogen that causes septicemia and polyserositis in ducks, is largely unknown. Here, the functions of the putative TonB-dependent iron transporter of RA-CH-1, B739_1343, in iron utilization and pathogenicity were investigated. Under iron-starved conditions, the mutant strain RA-CH-1ΔB739_1343 exhibited more seriously impaired growth than the wild-type strain RA-CH-1, and the expression of B739_1343 in the mutant strain restored growth. qRT-PCR results showed that the transcription of B739_1343 was not regulated by iron conditions. In an animal model, the median lethal dose (LD50) of the mutant strain RA-CH-1ΔB739_1343 increased more than 104-fold (1.6×1012 CFU) compared to that of the wild-type strain RA-CH-1 (1.43×108 CFU). In a duck co-infection model, the mutant strain RA-CH-1ΔB739_1343 was outcompeted by the wild-type RA-CH-1 in the blood, liver and brain of infected ducks, indicating that B739_1343 is a virulence factor of RA-CH-1. Finally, immunization with live bacteria of the mutant strain RA-CH-1ΔB739_1343 protected 83.33% of ducks against a high-dose (100-fold LD50) challenge with the wild-type strain RA-CH-1, suggesting that the mutant strain RA-CH-1ΔB739_1343 could be further developed as a potential live attenuated vaccine candidate for the duck industry.