Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Abstract

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92±70.6074 vs 263.10±34.60 pg/mL, P<.05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.