vscN encodes a type III secretion system ATPase in Vibrio splendidus AJ01 that contributes to pathogenicity and Hop secretion

Highlights

• VscN, the T3SS ATPase of Vibrio splendidus, was cloned and characterized.

• ∆vscN strain showed significantly attenuated virulence characteristics.

• VscN was associated with the expression and secretion of a T3SS effector Hop.

Abstract

The type III secretion system (T3SS) is a protein secretory pathway that is commonly used to inject virulence factors into the host cells through the injectosome, which is dependent on ATPase catalyzing the unfolding of proteins. In this study, a gene designated vscN in Vibrio splendidus AJ01, which was predicted to encode the T3SS ATPase based on its sequence similarity to other T3SS ATPases, was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant VscN possessed ATPase activity, catalyzing the release of ADP and Pi from ATP with an ATPase activity of 5.86 U/mg in a time- and dose-dependent manner. To determine whether VscN contributes to the pathogenesis of V. splendidus, we first constructed a mutant, ∆vscN, by creating an in-frame deletion in the vscN gene. ∆vscN showed no difference in growth compared with the wild-type strain, but in an immersion infection test, the survival rate of Apostichopus japonicus increased from 10% to 50% after challenge with the wild-type strain and the ∆vscN strain at a level of 5 × 10⁷ CFU/mL, respectively. After coincubation of coelomocyte with the wild-type strain or ∆vscN strain for 3 h, it was found that, in contrast to the wild-type strain, the ∆vscN strain negligibly accumulated at the cell surface of coelomocyte. In our previous study, Hop was found to be a T3SS effector, thus, fluorescence in situ hybridization and real-time reverse transcription PCR were performed to further show that the expression of the hop gene and the secretion of Hop were attenuated in the ∆vscN mutant strain. The results not only indicated that VscN is a T3SS ATPase that is involved in the virulence of AJ01 but also confirmed that Hop is a T3SS effector whose expression and secretion depend on VscN.

Introduction

As one of the most important economic aquaculture species in Northern China, the sea cucumber (Apostichopus japonicus, Echinodermata, and Holothuroidea) suffers from diseases such as skin ulceration syndrome (SUS), which is caused by bacterial infections due to high culture density in various culture modes (Wang et al., 2009; Liu et al., 2010; Han et al., 2016). Vibrio splendidus in particular is considered to be a main opportunistic pathogen that causes SUS in A. japonicus (Zhang et al., 2006). To date, virulence factors of metalloprotease Vsm, secretory protein Hop and hemolysin Vshppd have already been determined in V. splendidus, and each separately induces the cytotoxicity of A. japonicus coelomocytes (Liang et al., 2016; Zhang et al., 2016a; Zhuang et al., 2020). Vshppd, Hop and indole may also induce immune responses, including the affaction of oxidation-reduction states and the apoptosis of A. japonicus cells (Liang et al., 2016; Zhang et al., 2017). However, other virulence related factors await to be explored for understanding the overall pathogenesis of V. splendidus and for developing the ecological control methods.

The type III secretion system (T3SS) is used by many Gram-negative bacteria and contributes to bacterial pathogenesis through its ability to directly inject its bacterial cytoplasmic virulence proteins into host cells (Cornelis and Van Gijsegem, 2000; Macnab, 2004). Generally, more than 20 proteins are involved in the T3SS, which assemble into a complicated macromolecular structure with the ability to span the bacterial cell envelope (Blocker et al., 2003). In the V. splendidus AJ01 strain, the genomic sequence also includes T3SS components, including an ATPase, apparatus protein, inner-membrane P protein, exporter, and putative effector proteins (unpublished data). In T3SS, the chaperone-effector complex is recognized by the T3SS ATPase, after which binding between the ATPase and the complex occurs, followed by the dissociation of the chaperone-effector complex and the unfolding of effectors. The effectors are released into the cytoplasmic space at the tip of the injectosome that is inserted into the eukaryotic cell, where the effectors refold to exert their specific functions (Wulff-Strobel et al., 2002). The T3SS ATPase not only plays a role as an energizer in the secretion process but also provides energy for the protein-protein interactions that deliver effectors in the early stage of infection (Akeda and Galán, 2004). For example, T3SS ATPase InvC induced the release of chaperones and the unfolding of effectors in Salmonella enterica (Minamino and Macnab, 2000; Minamino et al., 2000; Akeda and Galán, 2005). Deletion of the yscN gene encoding the ATPase inhibited the secretion of all Yops in Yersinia enterocolitica (Woestyn et al., 1994). Compared with other strains, there has been no study on the T3SS and its delivery of T3SS effectors in V. splendidus.

In this study, a gene encoding the T3SS ATPase of V. splendidus AJ01, designated vscN, was cloned and characterized. The vscN gene was expressed in Escherichia coli BL21 (DE3), and the ATPase activity of VscN was determined. To better understand the function of VscN in the pathogenesis of V. splendidus, an in-frame deletion of vscN was performed, and the physiology and pathogenicity of ∆vscN were determined. In our previous study, Hop, showing cytotoxicity, was postulated to be a T3SS effector (Zhuang et al., 2020), and thus, in the present study, the expression and location of Hop in the ∆vscN strain were further determined.