Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A₂ in the...

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A₂ (PLA₂) may act as a tool to eliminate B. burgdorferi, but particular PLA₂ genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA₂ (pAaPLA₂) homologs in the present study, of which three pAaPLA₂ had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA₂s. Protein structural analysis demonstrated that pAaPLA₂s primarily consisted of α-helices, β-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA₂ activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA₂ proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA₂-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA₂_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA₂ may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA₂ genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.