Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses

Abstract

Extracellular products (ECPs) of the pathogenic Vibrio aestuarianus 01/32 were previously reported to display lethality in Crassostrea gigas oysters and to cause morphological changes and immunosuppression in oyster hemocytes. To identify the source of this toxicity, biochemical and genetic approaches were developed. ECP protease activity and lethality were shown to be significantly reduced following incubation with metal chelators, suggesting the involvement of a zinc metalloprotease. An open reading frame of 1836 bp encoding a 611-aa metalloprotease (designated Vam) was identified. The deduced protein sequence showed high homology to other Vibrio metalloproteases reported to be involved in pathogenicity. To further confirm the role of this enzyme in ECP toxicity, a plasmid carrying the vam gene under the control of an araC-P_BAD expression cassette was transferred to a Vibrio splendidus related strain, LMG20012^T, previously characterized as non-pathogenic to oysters. Expression of Vam conferred a toxic phenotype to LMG20012^T ECPs in vivo and cytotoxicity to oyster hemocytes in vitro. Collectively, these data suggest that the Vam metalloprotease is a major contributor to the toxicity induced by V. aestuarianus ECPs and is involved in the impairment of oyster hemocyte functions.

Introduction

Vibrio aestuarianus is a naturally occurring gram-negative bacterium, widely spread in marine ecosystems [1]. Recent epidemiological studies conducted during recurrent summer mortality events of Crassostrea gigas oysters along the French Atlantic coast have also documented the predominance of this bacterial species in the hemolymph of diseased animals, and have demonstrated its pathogenicity to C. gigas by experimental challenge [2], [3], [4]. Previous studies designed to understand V. aestuarianus pathogenicity mechanisms have shown that one of the isolated strains, named 01/32, secretes extracellular products (ECPs) which induce immunosuppressant activities on C. gigas hemocyte functions in vitro and display lethality to oysters in vivo [5]. During the time course of infection, this bacterial isolate was also reported to circumvent the host cellular immune defenses [6]. However, the mechanisms and bacterial effector(s) responsible for these immunomodulatory and toxic effects remain poorly understood. Since we previously established that V. aestuarianus 01/32 releases bacterial proteases into the host hemolymph during infection, we hypothesized that these proteases may be responsible, either directly or indirectly, for some of the observed pathological signs. Indeed, the pathogenesis of Vibrionaceae associated with marine invertebrate infections has frequently been linked with the production of extracellular proteases [7], [8], [9].

To date, only two studies have genetically demonstrated the causal relationship between proteases and virulence in these Vibrio agents, thus fulfilling the molecular version of Koch’s postulates [10], [11]. The most common procedure to prove cause-effect relationships for suspected bacterial virulence factors relies on loss-of-function studies using reverse genetic methods. However, genetic tools are sometimes not available in bacterial species of environmental origin because of either inoperative or inefficient DNA transformation, poor DNA delivery or inefficient allelic exchange. For such cases, gene expression in a heterologous system constitutes a useful alternative. A potential difficulty with this approach is that genes from heterologous systems may have adverse effects on cell growth and viability when expressed in Escherichia coli [12]. We recently characterized a close phylogenetic neighbor of V. aestuarianus, belonging to the Vibrio splendidus polyphyletic group [13]. This [13] strain, named LMG20012^T and previously reported to be non-pathogenic to oysters, can be easily manipulated genetically and is devoid of any protease activity [14]. Considering all these features, the V. splendidus related strain LMG20012^T constitutes an excellent candidate for heterologous expression of V. aestuarianus proteases. In the present study, a biochemical approach allowed us to associate V. aestuarianus ECP protease activity and lethality to oysters with the involvement of a metalloprotease-like enzyme. After identification of this factor, we successfully used LMG20012^T to heterologously express the V. aestuarianus zinc metalloprotease and genetically demonstrate its role in toxicity to C. gigas and impairment of oyster immune cells.