The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas

Abstract

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 microM Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan-Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

“…Taken together, the data from the present study unravel the cellular and molecular mechanisms associated to the OmpUdependent virulence of LGP32 evidenced in our previous study (6). We propose a model of hemocyte invasion by LGP32 in which LGP32 is recognized by β-integrin at the surface of oyster hemocytes through an OmpU-specific opsonisation by CgEcSOD (Fig.…”

“…In these cells, fibronectin/ vitronectin serve as molecular bridges between the pathogen and the host RGD-recognizing αvβ3-integrin receptors. As previously shown for fibronectin (6,14), the major protein of oyster plasma, known as Cg-EcSOD, was shown here to specifically recognize OmpU. From this study, Cg-EcSOD mediates attachment to and invasion of hemocytes, serving as an opsonin that promotes phagocytosis of LGP32 (Fig.…”

“…This finding showed that LGP32 remains viable and culturable within oyster hemocytes. In contrast, the avirulent control LMG20012 T (6,28), which was also avidly engulfed, did not survive within hemocytes, with cfu counts dropping by 4 logs over time (Fig. 2A).…”

“…Recent studies have shown that it mediates host-Vibrio interactions, being involved in the resistance to antimicrobial peptides (AMPs) (12,13), the adherence to host cells (14), or pathogen/symbiont recognition (15). Similarly, in LGP32, OmpU participates to the resistance to oyster AMPs and displays adhesive properties (6). To date, little is known on the molecular basis of the OmpU-mediated host-Vibrio interactions.…”

“…However, up to now, little has been known about the route of infection and pathogenic processes of LGP32 (2,3). A metalloprotease has been associated with toxicity (4,5) and the outer membrane protein (OMP) OmpU was shown to be a major determinant of LGP32 virulence (6).…”