Differential Proteomic Analysis Of Nematode Surface Coat Proteins Under Bacterial Protease Stress

Bacterial extracellular proteases have been long proposed as key virulence factors involved in bacterial infection processes against nematode, and most of them belong to subtilisin-like serine proteases. Previous studies have shown that enzyme-mediated degradation against nematode cuticles can facilitate nematophagous microbes to penetrate host cuticles. In this paper, pathogenic roles of a bacterial pathogenic protease BLG4 from Brevibacillus laterosporus and stress mechanism of nematode hosts before the enzyme attacks were investigated. Understanding of the bacterial degradation processes and self-protection mechanisms of nematodes can provide a basis to develop new biological control strategies for plant-parasitic nematodes.Previous histopathological studies have ever showed the hydrolytic enzyme BLG4 from B. laterosporus plays an important role in bacterial infection process against nematodes. In our study, a protease-deficient mutant was first constructed and analyzed to further identify the role at the molecular level. The in vitro protease-deficient mutant was generated by introducing the integration vector with a 445-bp protease BLG4 fragment into B. laterosporus chromosomal target via homologous recombination. The BLG4-deficient mutant showed a significant drop in protease activity as compared to the wild type G4 strain, but had a slight effect on bacterial growth and sporulation. Furthermore, the relationship between the activity of extracellular hydrolytic enzyme BLG4 and lethality of nematode target C.elegans was also observed and recorded. The obtained data showed that the activity of protease BLG4 was closely related lethality rate of nematode C.elegans. The 24h LC50 values of protease BLG4 against C.elegans were 1.0694μg/mL. Biochemical analysis and microscopic observation results showed that the cuticles of living nematodes (C.elegans) and dead ones exists an obvious difference in resistance to degradation of protease BLG4. These were consistent with provious histogenic immunity studies that different binding capacity of enzymes existed on different parts of nematode surface. The results suggested nematodes have self-protection mechanisms to resist degradation of hydrolytic enzymes.Suface coat is the outermost layer of nematode cuticle, and many studies have indicated that the surface coat displays dynamic properties thought to be involved in immune evasion. Thus, identification and analysis surface coat proteins (SCPs) of nematode cuticles and studying interaction mechanism between SCPs and protease BLG4 has important significance for exploring the mechanisms of enzyme degradation against nematode cuticle and self-protection mechanisms of nematode hosts. With C.elegans as the target material, we compared several methods for extracting nematode surface coat proteins to determine their efficacy in separating nematode SCPs by SDS-PAGE and 2D-PAGE. The results suggest that 35% ethanol method combined with TCA/acetone precipitation was the most effective step. And the SCPs samples were separated by 2-DE using 11cm pH 3-10 IPG Strip, isoelectric focusing(IEF) for 4000Vh and 12% SDS-PAGE. On the basis of this, we used differential proteomics approach to study the differences of protein expresion between 0.05μg/mL protease BLG4 stress and normal control. Finally,28 difference expression of spots were identified by using MALDI-TOF/TOF analysis. Among 16 of them were highly matched with the proteins pots from Maseot database by searching and intercomparison preliminary identifieation. These up- or down- proteins have close connection with nematode defense response, energy metabolism, biosynthesis, and so on. Future further researching those proteins could provided theoretical basis for elucidate molecular mechanisms of hydrolytic enzymes degradation on nematodes.