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

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 (triangle tolCA) but not the tolCB deletion mutant (triangle tolCB) was more sensitive to the NaOCl than the wild type (WT). Strikingly, the triangle tolCA but not triangle tolCB decreased the frequency of clones resistant to several tested antibiotics. In the duck infection model, the triangle tolCA but not triangle tolCB decreased virulence and colonization of R. anatipestifer CH-1, possibly due to increased sensitivity of triangle 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.