Identification of bacterial and viral proteins interacting with penaeus monodon antilipopolysaccharide factor isoform 3

A broad-spectrum antimicrobial peptide from Penaeus monodon, namely anti-lipopolysaccharide factor isoform 3 (ALFPm3) is active against bacteria, fungi and a shrimp pathogenic virus, white spot syndrome virus (WSSV). To study in depth on ALFPm3 function in shrimp immune responses and on ALFPm3-pathogen interaction, we screened the Vibrio harveyi (VHH)- and WSSV (WH)-infected shrimp hemocytes, V. harveyi (VH) and WSSV libraries for ALFPm3 interacting protein using yeast two-hybrid technique (Y2H). Screening of the bacterial- and viral-infected shrimp hemocyte libraries could not identify any ALFPm3-interacting partners. These suggested that the ALFPm3 did not interact with other shrimp proteins during pathogenic infection, implying that the ALFPm3 solely acted on the pathogens. Accordingly, no positive clone from VH library was found to interact with ALFPm3 suggesting that the antibacterial mechanism of ALFPm3 against V. harveyi might not involve its binding to bacterial proteins. For WSSV library, five true positive clones including WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471 were found to be the ALFPm3-interaction proteins. Temporal transcriptional analysis in WSSV-infected P. monodon hemocytes revealed that all WSSV genes were expressed in the late phase of infection (24 h and 48 h post infection). Then WSSV189 and WSSV471, an unknown protein, were selected for further analysis. The open reading frame (ORF) of each WSSV189 and WSSV471was cloned into pET-19b vector and subsequently transformed into different strains of Escherichia coli including BL-21(DE3), Rosetta(DE3)pLysS, and BL21-CodonPlus(DE3)-RIL. The recombinant proteins containing a His-tag at C-terminus of WSSV189 (rWSSV189) and WSSV471 (rWSSV471) were successfully expressed in E. coli strain BL21-CodonPlus(DE3)-RIL. The rWSSV189 (26 kDa) and rWSSV471 (20 kDa) were purified through Ni mediated affinity chromatography and checked by SDS-PAGE and Western blot analysis. In vitro pull-down assay using rWSSV189 and rWSSV471 as baits confirmed the true interaction between ALFPm3 with both WSSV proteins. To our knowledge, the specific binding of ALFPm3 to WSSV189 and WSSV471 might involve in anti-WSSV activity of ALFPm3.