Functional characterization of two TolC in the resistance to drugs and metals and in the virulence of Riemerella anatipestifer

Abstract

ABSTRACT TolC is an outer membrane protein that is responsible for the efflux of multi-substrates in Gram-negative bacteria. Riemerella anatipestifer CH-1 encodes two TolC proteins, B739_0871 (TolCA) and B739_1784 (TolCB). TolCB was previously involved in the export of manganese (Mn). However, whether TolCB has other substrates is unclear, and the functions of TolCA remain to be characterized. Here, we show that deletion of tolCA renders the bacteria hypersensitive to the gentamicin, the SDS, and various metals, such as Mn(II), Zn(II), Cu(II), Fe(II), Co(II), and Ni(II). By contrast, the deletion of tolCB only conferred high sensitivity to Mn. Consistently, the deletion of tolCA led to intracellular accumulation of the various tested metals, while the deletion of tolCB only led to Mn accumulation under conditions of excess Mn. Moreover, the tolCA deletion mutant (∆ tolCA ) but not the tolCB deletion mutant (∆ tolCB ) was more sensitive to the NaOCl than the wild type (WT). Strikingly, the ∆ tolCA but not ∆ tolCB decreased the frequency of clones resistant to several tested antibiotics. In the duck infection model, the ∆ tolCA but not ∆ tolCB decreased virulence and colonization of R. anatipestifer CH-1, possibly due to increased sensitivity of ∆ tolCA to non-inactivated duck serum as compared to the WT. Finally, genome mining revealed that homologs of tolCA and tolCB are highly conserved across R. anatipestifer strains and also present in other members of Flavobacteriaceae . Taken together, our results revealed that compared with TolCB, TolCA has a wider range of efflux substrates and functions in R. anatipestifer CH-1. IMPORTANCE Riemerella anatipestifer (RA) is a notorious duck pathogen, characterized by a multitude of serotypes that exhibit no cross-reaction with one another. Moreover, RA is resistant to various antibacterial agents. Consequently, understanding the mechanisms behind resistance and identifying potential targets for drug development have become pressing needs. In this study, we show that the two TolC proteins play a role in the resistance to different drugs and metals and in the virulence. The results suggest that TolCA has a wider range of efflux substrates than TolCB. Except for gentamicin, neither TolCA nor TolCB was involved in the efflux of the other tested antibiotics. Strikingly, TolCA but not TolCB enhanced the frequency of resistance-conferring mutations. Moreover, TolCA was involved in RA virulence. Given its conservation in RA, TolCA has potential as a drug target for the development of therapeutics against RA infections.