Chitosan oligosaccharide efficiently inhibits Cronobacter sakazakii biofilm by interacting with out membrane protein A for regulating CpxRA-mediated cellulose production pathway

Abstract

Chitosan oligosaccharide (COS) can efficiently inhibit Cronobacter sakazakii (C. sakazakii) biofilm independent on antibacterial activity. However, the mechanism is still unclear. In this study, the role of out membrane protein A (OmpA) and its downstream CpxRA-mediated cellulose production pathway in COS's inhibition on C. sakazakii biofilm were explored. The spectroscopic results were shown that COS could interact with OmpA, and this changed OmpA's second structure and spatial conformation as well as cell membrane permeability and COS uptake. C. sakazakii ΔOmpA strain under COS treatment had a lower cell membrane permeability and COS uptake rate. The interaction between OmpA and COS could further initiate CpxRA system. The regulon cpxP expression level was therefore up-regulated. The deletion of the response regulator cpxR gene reduced inhibitory effect of COS on biofilm. CpxRA system inhibited expression of csgD and adrA, which coded diguanylate cyclase to generate cyclic diguanosine monophosphate (c-di-GMP). The expression of bcsAB was then down-regulated by c-di-GMP, and the cellulose production as well as biofilm were reduced. The addition of exogenous c-di-GMP could mitigate the inhibition of COS on C. sakazakii biofilm. These results not only help to elucidate biofilm inhibition mechanism of COS, but also provided a basis for developing anti-biofilm agents targeted OmpA.