Immune signaling of Litopenaeus vannamei c-type lysozyme and its role during microsporidian Enterocytozoon hepatopenaei (EHP) infection

The microsporidian Enterocytozoon hepatopenaei (EHP) is a fungi-related, spore-forming parasite. EHP infection causes growth retardation and size variation in shrimp, resulting in severe economic losses. Studies on shrimp immune response have shown that several antimicrobial peptides (AMPs) were upregulated upon EHP infection. Among those highly upregulated AMPs is c-type lysozyme ( Lv Lyz-c). However, the immune signaling pathway responsible for Lv Lyz-c production in shrimp as well as its function against the EHP infection are still poorly understood. Here, we characterized major shrimp immune signaling pathways and found that Toll and JAK/STAT pathways were up-regulated upon EHP infection. Knocking down of a Domeless ( DOME ) receptor in the JAK/STAT pathways resulted in a significant reduction of the Lv Lyz-c and the elevation of EHP copy number. We further elucidated the function of Lv Lyz-c by heterologously expressing a recombinant Lv Lyz-c (r Lv Lyz-c) in an Escherichia coli . r Lv Lyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus . Interestingly, we found an antifungal activity of r Lv Lyz-c against Candida albican , which led us to further investigate the effects of r Lv Lyz-c on EHP spores. Incubation of the EHP spores with r Lv Lyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that r Lv Lyz-c possibly digest a chitin coat on the EHP spores. Transmission electron microscopy analysis revealed that an endospore layer, which is composed mainly of chitin, was digested by r Lv Lyz-c. Lastly, we observed that EHP spores that were treated with r Lv Lyz-c showed a significant reduction of the spore germination rate. We hypothesize that thinning of the endospore of EHP would result in altered permeability, hence affecting spore germination. This work provides insights into shrimp immune signaling pathways responsible for Lv Lyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.