Antibacterial activity and gene regulation of antilipopolysaccharidefactor from black tiger shrimp Penaeus monodon

Abstract

Antilipopolysaccharide factor (ALF) is one of the antimicrobial peptide families identified in invertebrates. Six different isoforms of the ALF homologues have been identified from Penaeus monodon. Previously, ALFPm6 has been shown to possibly play an important role in shrimp immunity against pathogen invasions. To further characterize ALFPm6, the nucleotide sequences coding for mature peptide of ALFPm6 were cloned and expressed in Pichia pastoris strain KM71. The recombinant ALFPm6 protein (rALFPm6) with the expected size and pI of 12 kDa and 9.69, respectively, was successfully produced. The crude rALFPm6 protein showed antibacterial activities against both Gram-positive and Gram-negative bacteria, such as Bacillus megaterium and Escherichia coli 363, respectively. Because of the unsuccessful rALFPm6 purification, the synthetic cyclic ALFPm6#29-52 peptide (cALFPm6#29-52) corresponding to ALFPm6 LPS-binding site was synthesized and analyzed for antibacterial activity. It exhibited the growth inhibition activity against some tested bacteria such as a Gram-negative bacterium, E. coli 363 as well as Gram-negative bacteria, B. megaterium, Aerococcus viridans, and Micrococcus luteus, with MBC value of 25-50 µM. Bacterial agglutination assay indicated that cALFPm6#29-52 induced bacterial agglutination mediated bacterial killing. Herein, the regulation of ALFPm3 and ALFPm6 gene expression was also studied. The 5′-upstream sequences of ALFPm3 (1780 bp) and ALFPm6 (504 bp) genes were identified by genome walking technique. Sequence analysis of ALFPm3 and ALFPm6 promoters identified several putative transcription factor (TF)-binding sites. Using narrow down assay, ALFPm3 and ALFPm6-promoter active regions located at nucleotide position (-814/+302) and (-282/+85), respectively, were identified. ALFPm3 promoter active region contained TF-binding sites of SP-1, ICSBP, and NF-κB plus 21 units of GAAAGAGAGTAAGAG[T/C] tandem repeat. ALFPm6 promoter active region contained TF-binding sites of SP-1, ICSBP, Oct-1, and C/EBPß. Afterthat, the activator-binding sites from -814 to -266 of ALFPm3 promoter and -282 to -81 of ALFPm6 promoter were identified. Site-directed mutagenesis at the conserved nucleotide of selected TF-binding sites revealed that Rel/NF-κB binding site (-280/-270) and C/EBPß (-88/-78) binding site were the activator-binding site of ALFPm3 and ALFPm6 promoters, respectively. RNAi knockdown of MyD88 and Relish genes in Vibrio harveyi-infected shrimp suggested that ALFPm3 gene expression might be regulated by both Toll and IMD pathways, while ALFPm6 gene expression might be regulated by Toll pathway.