Abstract
Lysozyme is an enzyme that cleaves the β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, leading to bacterial lysis. Recently, lysozyme has been found to have anti-HIV and anti-cancer properties in mammals. However, most functional analyses were done in vitro using purified or recombinant lysozyme protein. Here, we used RNA interference to silence c-type lysozyme expression in penaeid shrimp, Marsupenaeus japonicus, to analyze the function of lysozyme in vivo. Silencing of lysozyme expression by dsRNA lysozyme (dsLYZ) led to 100% mortality without any artificial bacterial infection in 5 days. Lysozyme deficiency caused the number of hemocytes in hemolymph to decrease from 1.3 × 10(7) to 2.3 × 10(6) cells/ml and caused the number of bacteria to increase from 78 to 764 colony-forming units/ml. Suppression of bacterial growth using oxytetracycline and kanamycin showed improvement in mortality, suggesting that shrimp mortality post- dsLYZ injection can be attributed to bacterial growth in the shrimp hemolymph. The majority of the bacteria, identified by 16 S rRNA analysis, were Gram-negative species such as Vibrio and Pseudomonas. Furthermore, PKH26 staining showed that the dsLYZ-injected shrimp were unable to eliminate non pathogenic Escherichia coli or Staphylococcus aureus in 24 h. These data suggest that c-type lysozyme in shrimp serves to regulate the growth of bacterial communities, particularly Gram-negative bacteria, in the hemolymph.
Highlights
Lysozyme is ubiquitous in many organisms and functions primarily in bacterial lysis
Suppression of bacterial growth using oxytetracycline and kanamycin showed improvement in mortality, suggesting that shrimp mortality post- doublestranded RNA (dsRNA) lysozyme (dsLYZ) injection can be attributed to bacterial growth in the shrimp hemolymph
Western blot analysis showed that lysozyme protein was suppressed in the gills, hemocytes, and lymphoid organ at day 3 post-dsLYZ injection (Fig. 1E)
Summary
Lysozyme is ubiquitous in many organisms and functions primarily in bacterial lysis. Results: The absence of lysozyme in shrimp resulted in the increase of bacteria in the hemolymph leading to mortality. PKH26 staining showed that the dsLYZ-injected shrimp were unable to eliminate non pathogenic Escherichia coli or Staphylococcus aureus in 24 h These data suggest that c-type lysozyme in shrimp serves to regulate the growth of bacterial communities, Gram-negative bacteria, in the hemolymph. AMPs are small cationic molecules that form the first line of host defense against pathogenic infections; they possess potent antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, parasites, and some viruses [2,3,4]. Lysozyme is a bacteriolytic enzyme that is found in both prokaryotes and eukaryotes It mainly hydrolyzes the -1, 4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan leading to bacterial lysis [25]. Our results on the localization and anti-bacterial function of lysozyme confirm that it has an important role in crustacean immunity
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