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

Abstract

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 (LvLyz-c). However, the immune signaling pathway responsible for LvLyz-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 LvLyz-c and the elevation of EHP copy number. We further elucidated the function of LvLyz-c by heterologously expressing a recombinant LvLyz-c (rLvLyz-c) in an Escherichia coli. rLvLyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus. Interestingly, we found an antifungal activity of rLvLyz-c against Candida albican, which led us to further investigate the effects of rLvLyz-c on EHP spores. Incubation of the EHP spores with rLvLyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that rLvLyz-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 rLvLyz-c. Lastly, we observed that EHP spores that were treated with rLvLyz-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 LvLyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.

Fig 1. Temporal expression of the immune-related genes upon EHP infection.

(A-C), JAK/STAT pathway; (D-F), Toll like receptor (TLR) pathway; (G-I), Antimicrobial peptides (AMPs). Shrimp were reared together with EHP-positive shrimps. Immune-related gene transcriptional levels were determined via qRT-PCR at 1, 9, 11, and 15 days after EHP cohabitation. Each data point was normalized to the expression of the EF-1α gene and calculated relative to the expression level in the specific pathogen-free (SPF) shrimp. Dotted line represents the expression level in the SPF shrimp. Error bars represent the standard deviation of three biological replicates (n = 3). All the data were analyzed by one-way ANOVA. * represents P<0.05, ** shows P<0.01, and *** indicates P<0.001.

Fig 2. Effect of the immune-related receptor knockdown on LvLyz-c expression level and EHP copy number.

(A-C) Domeless receptor and (D-F) Toll-like receptor 2 (TLR2) knockdowns. The mRNA expression level of LvDOME, LvTLR2, and LvLyz-c were quantified by qRT-PCR. EHP copy numbers were analyzed using absolute qPCR comparing with aSSU rRNA gene of EHP. The GFP dsRNA treated shrimp were used as a control. All the data were analyzed by one-way ANOVA. bars with * indicate statistically significant differences (P<0.05); bars with ** indicate highly statistically significant (P<0.01).

Fig 3. Luciferase assay of LvSTAT and LvLyz-c promoter transiently expressed in the HEK293 cells.

(A) Schematics of a luciferase reporter gene and constructs of protein expression plasmids. ΔSBS represents a construct where the STAT binding site (10 bp) was removed. (B) Transient luciferase assay detection. Values are means ± standard errors of three independent replicates (n = 3). Statistical significance was determined by student T-test (** P < 0.01).

Fig 4. Agar diffusion antimicrobial assay of a recombinant LvLyz-c.

(A) Antimicrobial plate assay against gram negative bacteria Vibrio parahaemolyticus, gram positive bacteria Bacillus subtilis, and yeast Candida albican. (B) Clear zone inhibition measurement shown in millimeter units. Values are means ± standard errors of three independent replicates (n = 3).

Fig 5. Enzymatic digestion of the EHP endospore layer.

(A) Fluorescent micrographs of EHP spores stained with a chitin dye. (B) Quantification of the fluorescence intensity of EHP spores from (A). (C) Representative TEM micrographs. Red arrows represent the endospore layer. (D) Quantification of the EHP spore wall thickness and (E) spore wall area. Each experiment was performed in three biological replicates (n = 100 for each replicate, except in (E) that n = 10). ** P<0.01 and *** (P<0.001. Scale bars for the micrographs are 5 μm for fluorescence analysis and 200 μm for TEM.

Fig 6. Inhibitory effect of rLvLyz-c on EHP spore germination.

(A) EHP spore germination after incubating with rLvLyz-c (B) Quantification of the EHP spore germination rate. Each experiment was performed in three independent biological replicates (n = 100 for each replicate). ** P<0.01 and *** P<0.001. Scale bar is 5 μm.

Fig 7. Schematic representation of EHP-mediated activation of the Lyz-c production by TLR2 or JAK-STAT signaling pathway.

EHP induces TLR2 and DOME receptors to activate their downstream effectors, LvMyD88 and LvJAK, respectively. LvDorsal or LvSTAT transcription factor promotes a specific set of antimicrobial peptides, including LvLyz-c, LvPEN3, and LvALF1. LvLyz-c production upon the infection could digest the endospore layer of EHP and inhibit the EHP germination process. Note that Fig 7 was created with BioRender.