ALFPm6, a member of antimicrobial peptide in the antilipopolysaccharide factor (ALF) family from Penaeus monodon, plays important roles in shrimp immunity against pathogens. However, its antimicrobial activity and underlying mechanism have not been reported. The synthetic cyclic ALFPm6#29-52 peptide (cALFPm6#29-52) corresponding to the ALFPm6 LPS-binding domain can agglutinate and exhibited bacterial killing activity toward a Gram-negative bacterium, Escherichia coli 363 and Gram-positive bacteria, Bacillus megaterium, Aerococcus viridans, and Micrococcus luteus, with MIC values of 25-50 μM. Specifically, ALFPm6 and ALFPm3, the most abundant ALF isoforms, are different in terms of gene expression patterns upon pathogen infections. Herein, the regulation of ALFPm3 and ALFPm6 gene expression was studied. The 5'-upstream and promoter sequences were identified and the putative transcription factor (TF)-binding sites were predicted. The narrow down assay indicated that the ALFPm3 promoter and partial promoter of the ALFPm6 active regions were located at nucleotide positions (-814/+302) and (-282/+85), respectively. Mutagenesis of selected TF-binding sites revealed that Rel/NF-κB (-280/-270) of ALFPm3 and C/EBPβ (-88/-78) and Sp1 (-249/-238) sites of ALFPm6 were the activator-binding sites. Knockdown of the PmMyD88 and PmRelish genes in V. harveyi-infected shrimp suggested that the ALFPm3 gene was regulated by Toll and IMD pathways, while the ALFPm6 gene was regulated by the Toll pathway.

中文翻译：

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斑节对虾中抗脂多糖因子ALFPm3和ALFPm6的调节。

ALFPm6是斑节对虾的抗脂多糖因子（ALF）家族中抗菌肽的成员，在虾对病原体的免疫中起重要作用。但是，尚未报道其抗菌活性和潜在机理。对应于ALFPm6 LPS结合结构域的合成环状ALFPm6＃29-52肽（cALFPm6＃29-52）可以凝集并显示出对革兰氏阴性细菌，大肠杆菌363和革兰氏阳性细菌，巨大芽孢杆菌，绿色气单胞菌和黄褐微球菌，MIC值为25-50μM。具体而言，ALFPm6和ALFPm3是最丰富的ALF亚型，在病原体感染后的基因表达方式方面有所不同。在此，研究了ALFPm3和ALFPm6基因表达的调控。5' 上游和启动子序列进行了鉴定，并预测了假定的转录因子（TF）结合位点。缩小测定表明ALFPm6活性区的ALFPm3启动子和部分启动子分别位于核苷酸位置（-814 / + 302）和（-282 / + 85）。选定的TF结合位点的诱变表明，ALFPm3的Rel /NF-κB（-280 / -270）和ALFPm6的C /EBPβ（-88 / -78）和Sp1（-249 / -238）位点是激活剂-结合位点。harveyi感染虾中PmMyD88和PmRelish基因的敲低表明，ALFPm3基因受Toll和IMD途径调控，而ALFPm6基因受Toll途径调控。缩小测定表明ALFPm6活性区的ALFPm3启动子和部分启动子分别位于核苷酸位置（-814 / + 302）和（-282 / + 85）。选定的TF结合位点的诱变表明，ALFPm3的Rel /NF-κB（-280 / -270）和ALFPm6的C /EBPβ（-88 / -78）和Sp1（-249 / -238）位点是激活剂-结合位点。harveyi感染虾中PmMyD88和PmRelish基因的敲低表明，ALFPm3基因受Toll和IMD途径调控，而ALFPm6基因受Toll途径调控。缩小测定表明ALFPm6活性区的ALFPm3启动子和部分启动子分别位于核苷酸位置（-814 / + 302）和（-282 / + 85）。选定的TF结合位点的诱变表明，ALFPm3的Rel /NF-κB（-280 / -270）和ALFPm6的C /EBPβ（-88 / -78）和Sp1（-249 / -238）位点是激活剂-结合位点。harveyi感染虾中PmMyD88和PmRelish基因的敲低表明，ALFPm3基因受Toll和IMD途径调控，而ALFPm6基因受Toll途径调控。