Analysis of Secreted Proteins and Potential Virulence via the ICEs-Mediated Pathway of the Foodborne Pathogen Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus uses bacterial secretion systems and integrative and conjugative elements (ICEs) to induce various diseases and to adapt to harsh environments, respectively. Information pertaining to the identity of secreted proteins and functional characterization of ICEs has been previously reported, but the relationship between these elements remains unclear. Herein we investigated secreted proteins of V. parahaemolyticus strains JHY20 and JHY20△ICE using two-dimensional gel electrophoresis and LC-MS/MS, which led to the identification of an ICE-associated secreted protein - dihydrolipoamide dehydrogenase (DLDH). Considering the data related to its physical and biochemical characterization, we predicted that DLDH is a novel immunogenic protein and associated with virulence in JHY20. Our findings indicate a potential relationship between ICE-associated transport and secreted proteins and shed light on the function of such transport mechanisms. We believe that our data should enhance our understanding of mobile genetic elements.

Keywords: Vibrio parahaemolyticus; bacterial secretion systems; dihydrolipoamide dehydrogenase; integrative and conjugative elements; secreted protein.

Figure 1

SDS-PAGE analysis of the Vibrio parahaemolyticus extracellular products. Protein molecular weight marker (Tiangen, Cat. No. DPP530S) (Lane 1), an extracellular product of Vibrio parahaemolyticus JHY20△ICE (Lane 2), and Vibrio parahaemolyticus JHY20 (Lane 3) cultured under the same condition at 37°C for 12h.

Figure 2

Identification of secreted protein by two-dimensional gel electrophoresis (2-DE). Discrimination of secreted proteins between JHY20 (A) and JHY20△ICE (B). Twelve discrepancy protein spots were detected in the supernatant of strain, indicated by numbers in red.

Figure 3

b and y ions annotation of MS/MS fragmentation of IWDSTDALELK.

Figure 4

MS and MS/MS spectrum of IWDSTDALELK. (A) MS spectrum of IWDSTDALELK. (B) MS/MS spectrum of IWDSTDALELK.

Figure 5

A multiple sequence alignment of Vibrio parahaemolyticus strain JHY20 dihydrolipoamide dehydrogenase (DLDH) and a selected set of bacterial DLDH proteins. Numbers above the alignments indicate relative positions of the entirely aligned sequences. Identical amino acid residues, as well as the conserved ones (>50% of the sequences), are highlighted in black and in gray, respectively. The FAD and pyridine nucleotide-binding motifs (GXGXXG) are in red box. Two catalytic cysteine residues (GGXCXXXGCXP) and His-Glu pair residues (HXXXXE) are indicated by a green box and blue box, respectively. The DLDH sequences chosen for the analysis are obtained from GenBank and NCBI databases: GI: 491522365: Vibrio harveyi; GI: 484573283: Vibrio alginolyticus; GI: 31541532: Mycoplasma gallisepticum str. R(low); GI: 512523: N. meningitides serogroup B; GI: 482677317: Escherichia coli; GI: 589917356: Aeromonas caviae; GI: 27467712: Staphylococcus epidermidis ATCC12228. JHY20_DLDH, obtained in this study.

Figure 6

Phylogenetic tree showing evolutionary relationship between the Vibrio sp. DLDH and some characterized bacterial DLDH and related proteins. Based on the DLDH amino acid sequences derived from JHY20 in this study and from some known DLDH in the public databases, the neighbor-joining phylogenetic tree was constructed by using the MEGA 6.06. Bootstrap percentages are shown at nodes. The scale bar represents 0.1 changes per amino acid. Filled cycles denote the bacterial DLDH proteins that have been characterized for their activity or function, whereas JHY20 DLDH identified in this study is marked with a solid triangle.