Identification of genes for antimicrobial effectors and characterization of anti-lipopolysaccharide factor of the black tiger shrimp Penaeus monodon

Abstract

Differential Display PCR technique (DD-PCR) was used for the analysis of altered gene expression in haemocytes of Vibrio harveyi infected Penaeus monodon. Forty-four combinations of arbitrary and oligo-dT primer were used to screen for differentially expressed genes. A total of 79 differentially expessed bands could be identified from 33 primer combinations. These included 48 bands (61%) whose expression level increased and 31 bands (39%) decreased in expression after V. harveyi challenge Forty-eight differential display fragments were successfully reamplified and cloned. These clones were randomly selected and partially sequenced. The sequence analysis showed that 21 (44%) out of 48 bands contained unique sequences, while the remaining bands contained mixture of two to four different sequences. Eighty-five (31%) out of 267 clones matched with sequences in the GenBank database which represented 24 different genes. Caspase 3B, SERPINB3, profilin, lysozyme, glucose transporter 1, interferon-related developmental regulator 1, were selected for further confirmation for their differentially expression patterns by real-time PCR. The results indicated that all selected genes were up-regulated in P. monodon haemocytes upon V. harveyi challenge. Anti-lipopolysacharide factor type3 (ALFPm3) cDNAs, an antimicrobial peptide, previously identified as the most abundant type of ALF in P. monodon was the only one type of ALF identified in this study. In order to characterize their biological activity and properties, we expressed the cDNA of ALFPm3 in yeast Pichia pastroris. The purified protein exhibited a strong activity against several strains of Gram-negative and -positive bacteria and fungi. Analysis of the antibacterial action against tested strains, Bacillus megaterium and Escherichia coli 363, suggested that the inhibition was due to bacteriocidal not bacteriostatic effect. The spatio-temporal expression of ALFPm3 encoding gene was studied in response to V. harveyi challenge by in situ hybridization and real-time PCR analysis. ALFPm3 expression was significantly increased in haemocytes at the early phase (6 hr) of microbial infection. Localization of ALFPm3 transcripts in shrimp tissues revealed that bacterial invasion resulted in distribution of ALFPm3 expressing haemocytes throughout the shrimp cepharothorax during the first phase of infection. The ALFPm3 transcripts reached high level of expression again at 72 hour-post injection. During the first phase of immune response, migraion of ALFPm3 producing haemocytes to the injection site was evidenced, when detection of ALFPm3 protein with a specific antibody was performed. Taken together, these results implied the crucial role of this effector in protection of shrimp against microbes.