Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

“…However, identifying the pathogenic serovars of V. parahaemolyticus by use of only these two toxins is not sufficiently accurate, as other hemolysins may take part in the pathogenicity of V. parahaemolyticus. Thermolabile hemolysin (TLH), a toxin encoded by the tlh gene of V. parahaemolyticus and present in almost every clinical and environmental V. parahaemolyticus strain (14, 37), has been suggested as a promising target for pathogen detection (30,35,44). Although TLH has hemolytic activity and can lyse red blood cells, its cytotoxic and biochemical mechanisms of action are still not clearly understood (3,26,31).…”

Vibrio Parahaemolyticus: A review on distribution, pathogenesis, virulence determinants and epidemiology

“…They have all been included in the tdh family because the coding sequences of these genes share >93% homology. [ 33 ] Clinical samples from an outbreak of gastroenteritis in the Republic of Maldives presented KP-negative strains of V. parahaemolyticus . However, these strains were found to express a new hemolysin, termed as TRH.…”

“…[ 29 ] It should not escape our notice that V. parahaemolyticus bacteria express an additional toxin, known as thermolabile hemolysin (TLH). [ 33 ] It is encoded by the tlh gene, which is targeted during the genetic detection of V. parahaemolyticus because it is a species-specific marker. [ 13 ] TLH exhibits phospholipase activity and the ability to lyse human erythrocytes.…”

“…[ 32 ] However, its direct contribution to the pathogenicity of V. parahaemolyticus is yet to be elucidated. [ 33 ]…”

Figure 4: Effect of phenolic acids on Vp TLH enzymatic activity.

“…Shinoda et al (1991) first reported the recombinant production of VpTLH as an active soluble protein from the periplasm of E. coli (Shinoda et al, 1991). Recombinant hemolysins TDH, TRH, and TLH from Vp also were expressed as inactive form and renatured by carbamide gradient dialysis (Zhao, Tang & Zhan, 2011). Despite these differences, recombinant VpTLH showed lecithin-dependent phospholipase and hemolytic activity as other Vibrio TLHs (Jia, Woo & Zhang, 2010;Li, Mou & Nelson, 2013;Zhao, Tang & Zhan, 2011).…”