Molecular cloning and expression profiles of chicken NK-lysin during Eimeria infections

Yeong H. Hong*, Hyun S. Lillehoj#,
Rami A. Dalloul, Kate B. Miska, Wenbin Tuo, and Sung H. Lee
Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705, USA

ABSTRACT
NK-lysin is known to have antibacterial and antitumor activities and is expressed by NK cells as well as T-lymphocytes. In a previous report, the contig 171 (NK-lysin-like sequence), composed of 87 ESTs, occurred with the highest prevalence in an Eimeria-induced intestinal cDNA library. This study was conducted to clone, express, and characterize contig 171 as the chicken NK-lysin, which consists of an 868 bp DNA sequence and an open reading frame of 140 amino acids with a predicted molecular mass of 15.2 kDa. Comparison of deduced amino acid sequences showed less than 20% identity to granulysin and other mammalian NK-lysins. When infected with E. maxima, chicken total intraepithelial lymphocytes (IELs), CD4+ and CD8+ IELs showed higher expression levels of NK-lysin mRNA than CD8- IELs as quantified by real-time PCR. Upon infections with three Eimeria species, chicken NK-lysin RNA expression level normalized to GAPDH exhibited differential and interesting patterns. Further, chicken NK-lysin was expressed into COS-7 cells and its antitumor activity was demonstrated in killing LSCC-RP9 macrophage transformed cells. Also, chicken NK-lysin showed sequence similarity to other mammalian cDNA with cytotoxic activity to tumor cells. In these studies, we cloned and characterized chicken NK-lysin and studied its expression patterns in mRNA of Eimeria-infected birds. As in other animals, chicken NK-lysin may play an important role in antimicrobial defenses against enteric pathogens such as Eimeria, especially at the early stages of such infections.


*Presenting Author
#Corresponding Author
E-mail: hlilleho@anri.barc.usda.gov