Down-Regulation of Lipid Metabolism in the Hepatopancreas of Shrimp Litopenaeus vannamei upon Light and Heavy Infection of Enterocytozoon hepatopenaei: A Comparative Proteomic Study

Abstract

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Keywords: Enterocytozoon hepatopenaei; Litopenaeus vannamei; lipid metabolism; proteomic.

Figure 1

Sample information and basic statistics of proteomic data. (A): Shrimps in the pathogenetic pond presented various sizes. The large ones reached 10 cm (blue dotted line), while the little ones were less than 8 cm (red dotted line). (B): Summary of differentially expressed proteins, and the up- and down-regulated numbers were represented with red and blue columns, respectively. (C): PCA analysis was used to assess the repeatability of samples. (D): Shrimp samples were divided into three groups based on EHP load quantified by EHPptp2 RT-qPCR. The EHP quantity of each group was more intuitive by fluorescence microscopy, as the fluorescent brightener 28 (FB28) could label the spore wall of EHP. Each blue, fluorescent spot represented an EHP spore.

Figure 2

The function of DEPs classified by GO and GOC. (A–C): Go classification revealed that proteins involved in the cellular metabolism process with binding or/and catalytic activity functions were regulated after EHP infection (green and purple columns). The length of the column represents the enriched proteins. The comparison of the results also suggested that the quantitative change of host proteins was related to the EHP level. (D–F): COG classification indicated that the regulated proteins in EHP-infected hepatopancreas mainly play a role in lipid and amino acid transport and metabolism processes (green columns with bold).

Figure 3

Functional enrichment clusters of differentially expressed proteins. KEGG enrichment of differentially expressed proteins. One color block represents a functional pathway of DEPs enrichment in different comparison groups, red represents a high degree of enrichment, and blue represents weak enrichment. The entire enriched KEGG pathways mainly manifested as down-regulated lipid and amino acid metabolism, and the regulation level between the H and L groups was discrepant.

Figure 4

Expression pattern of identified proteins and modification sites via Mfuzz analysis. Mfuzz analysis result of the proteome. The left broken line charts show the protein expression trends in each sample. The horizontal axis indicates different groups, the vertical axis indicates the protein expression amount, and one broken line represents one protein. According to the different trends of the broken lines, they were divided into six different trends through cluster analysis. Heat maps were drawn for the protein sets in each cluster. To further understand the biological processes in which proteins in each cluster are involved, we carried out an enrichment analysis of the KEGG pathway of proteins in each cluster. The first few of the most significant enrichment were listed on the right.

Figure 5

Summary of the main differentially regulated pathways between the H and L group. Green-colored ellipses, rectangular frames, and polygons indicate down-regulated pathways. In comparison, the orange-colored ones indicate up-regulated processing. Purple-colored ones indicate the participant enzymes or the fold enrichment value of metabolism pathways, and the value in brackets represents the ratio of H to L.

Figure 6

Transcription detection of target genes. Relative quantitation RT-qPCR verified the mRNA level of genes correlated with fatty acids, carbohydrates, hormones, and amino acid metabolism. The log₂ (relative ratio) of target genes was used to assess the condition of gene regulation. Different colored columns represent different comparative levels. LDH: lactate dehydrogenase; PDHE1β: pyruvate dehydrogenase E1 component subunit beta; PC: pyruvate carboxylase; ACACA: acetyl-CoA carboxylase; Lip1: Triacylglycerol lipase; CPT1: carnitine O-palmitoyltransferase 1; SCD: Acyl-CoA delta-9 desaturase; JHE1: JHE-like carboxylesterase 1; EcdR: Ecdysteroid regulated-like protein; SPN: pacifastin light chain-like serine proteinase inhibitor; PPO2: Prophenoloxidase-2; PSaT: phosphoserine aminotransferase; SSDHm: mitochondrial succinate-semialdehyde dehydrogenase.

Figure 7

Information of identified pathogen proteins in the H and L group. (A): Statistic data of EHP proteins. The outer ellipse represents 479 EHP proteins found in the H group, and the inner ellipse indicates 217 EHP proteins were found in the L group. (B): Predicted subcellular location of EHP proteins found in infected groups. Protein numbers were shown on or side of each item.

Figure 8

Overview of the main regulatory pathways in the EHP-infected hepatopancreas. The green color indicates pathways, and the arrows represent down-regulated metabolic processing and reaction steps. The red-colored arrow represents up-regulated reaction steps and orange colored indicates up-regulated processing or proteins. The purple color indicates the participant enzymes or the fold enrichment value of metabolic pathways, and the value in front of the diagonal represents the ratio of H vs. C, while the value behind the diagonal represents the ratio of L vs. C.