Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodon

Lysozyme is a widely distributed hydrolase possessing a hydrolytic activity against peptidoglycan in the bacterial cell wall and, hence, causing lysis of the bacteria. Two types of lysozymes; the c-type (PmLyzc) and the two catalytic residue ablated i-type lysozymes (PmLyzi1 and 2), were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th). By RT-PCR, PmLyzc transcript was detected in all tissues: gill, antennal gland, epipodite, heart, hemocyte, hepatopancreas, eyestalk, lymphoid organ and intestine, and highly expressed in hemocyte. The expression of PmLyzi2 mRNA was highest in heart while undetected in gill, lymphoid organ and intestine. The PmLyzi1 transcript was expressed only in hepatopancreas. The up-regulation of mRNA transcription after bacterial challenge was observed only with PmLyzc. To investigate their biological activities, the three mature recombinant proteins were expressed in an Escherichia coli system. Although the turbidimetric assay revealed that only recombinant PmLyzc possessed the muramidase activity, all of them variably exhibited antimicrobial activity against both Gram-positive and -negative bacteria especially the shrimp pathogens, Vibrio species. The antimicrobial activities of recombinant PmLyzc was the most effective one. These results demonstrated that the ability of lysozyme to inhibit the growth of bacteria did not depend only on the muramidase activity. Differences in tissue expression pattern of these gene transcripts and their antimicrobial activities indicated the multifunction of lysozyme as immune defense and digestive enzymes in P. monodon.