Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

Abstract

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation-reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

Keywords: Crassostrea gigas; cathepsin L; lysosome; oxidative killing; phagocyte; quantitative proteome; transcriptome.

Figure 1

Pipeline overview of transcriptome and proteome analysis of Crassostrea gigas phagocytes. The hemocytes are collected, incubated with FITC-labeled latex beads, and sorted using FACS to prepare both phagocytes and non-phagocytic hemocytes. Differential expression in phagocytes is revealed using both transcriptome and quantitative proteome analyses. A complex network is constructed based on differentially expressed proteins and further validated using qPCR, flow cytometry, and confocal microscopy.

Figure 2

Gene ontology enrichment analysis of differentially expressed genes (DEGs) from the transcriptome. Enrichment analysis results are presented here in the form of scatterplots of high (A) and low expressed (B) phagocyte DEGs. The enrichment factor is the ratio between the DEG number and the number of all genes in a certain gene enrichment term. The sizes of the dots on these plots denote the number of DEGs, while colors correspond to the q value range.

Figure 3

iTRAQ analysis of proteins differentially expressed in phagocytes. (A) This chart illustrates the overall protein expression level identified in phagocytes. The average log₂-fold change for each protein is plotted, while those identified as either significantly high or low expressed are highlighted in red and green, respectively. (B) Statistical analysis of high and low expressed phagocyte differentially expressed proteins.

Figure 4

Gene ontology enrichment analysis of differentially expressed proteins from iTRAQ. The results of this enrichment analysis are presented as scatterplots of high (A) and low expressed (B) phagocyte differentially expressed proteins (DEPs). The enrichment factor in this case is the ratio of the DEP number to the number of all proteins in a certain enrichment term; the dot size in this figure denotes the number of DEPs, while colors correspond to the q value range.

Figure 5

Venn-regional analysis of phagocyte differentially expressed genes and differentially expressed proteins. These diagrams illustrate overlap between significantly high (red) and low expressed (green) genes and proteins. Transcriptomic and quantitative proteomic analyses reveal a total of 4,992 genes and 352 proteins that are significantly high expressed in phagocytes (A), and 3,524 and 205 that are significantly low expressed, respectively (B). The percentages of overlapping high and low expressed molecules at messenger RNA and protein levels are indicated on this figure and are labeled with yellow and blue colors, respectively.

Figure 6

Modulation network of differentially expressed proteins (DEPs) in phagocytes. DEPs were analyzed using the software STRING and imported into Cytoscape for manual curation and grouping. The node colors on this figure correspond to absolute maximum log₂ ratio-of-ratios; significant increases and decreases in phagocyte DEPs are highlighted in red and blue, respectively.

Figure 7

Relative expression analysis of differentially expressed proteins (DEPs) using qPCR. The expression of significantly high expressed (A) and low expressed (B) proteins within phagocytes and non-phagocytic hemocytes was determined using qPCR. Results are indicated here at the messenger RNA level for each phagocyte gene in relation to their level in non-phagocytic hemocytes (n = 6), *p < 0.05, **p < 0.01.

Figure 8

Differential expression of cathepsin L in hemocytes. (A) In this case, rCathepsin L was expressed and the purified form was separated using SDS-PAGE followed by Coomassie brilliant blue staining (left box). Western blotting analysis was then performed using polyclonal IgG against rCathepsin L (right box). IB: immunoblot. (B) Histogram to show the relative expression level of cathepsin L in phagocytes (gray) and non-phagocytic hemocytes (black) determined by flow cytometry. (C) The mean fluorescence intensity of cathepsin L was statistically calculated; these results are expressed as means ± SEM (n = 5), **p < 0.01.

Figure 9

The involvement of cathepsin L in hemocyte phagocytosis of Vibrio splendidus. Hemocytes were incubated with FITC-labeled V. splendidus to allow hemocyte phagocytosis. Subsequent to cell fixation and permeabilization, hemocytes were stained with polyclonal IgG against rCathepsin L and then subjected to Alexa Fluor 594-labeled anti-mouse IgG antibody staining. diamidino-2-phenylindole (DAPI) was used to mark the cell nucleus, and each image was captured at a single focal plane using confocal microscopy. Interactions between cathepsin L and engulfed microbes are denoted here with white arrows; scale bar: 10 µm.