Hemocytes play unequivocally central roles in host immune defense of bivalve mollusks, though the exact mechanisms underlying their functional differentiation are only partially understood. To this end, granulocytes and hyalinocytes were sorted via flow cytometry from hemocytes of the Pacific oyster Crassostrea gigas, and consequently quantitative transcriptomic analysis revealed a striking array of differentially expressed genes (DEGs), which were globally upregulated in granulocytes, dedicating to functional differentiation among oyster hemocytes. Our network of DEGs illustrated actively engaged signaling pathways, with Cdc42/Cdc42l being a core regulator of pathway network, which was validated by a dramatically reduced capacity for hemocyte phagocytosis in the presence of Cdc42 inhibitors. Additionally, a number of transcription factors were identified among DEGs, including ELK, HELT, and Fos, which were predominantly expressed in granulocytes. The AP-1 transcription factor Fos was confirmed to facilitate functional differentiation of hemocytes in an assay on binding to target genes by the AP-1 binding site, consistent with downstream phagocytosis and ROS production. Importantly, Cdc42/Cdc42l were also regulated by the expression of Fos, providing a possible regulatory mechanism-guided hemocyte functional differentiation. Findings in this study have bridged a knowledge gap on the mechanistic underpinnings of functional differentiation of hemocytes in a marine invertebrate C. gigas, which promise to facilitate research on the evolution of immune defense and functional differentiation of phagocyte in higher-order and more recent phyla.

Vibrio species are ubiquitously distributed in marine environments, with important implications for emerging infectious diseases. However, relatively little is known about defensive strategies deployed by hosts against Vibrio pathogens of distinct virulence traits. Being an ecologically relevant host, the oyster Crassostrea hongkongensis can serve as an excellent model for elucidating mechanisms underlying host-Vibrio interactions. We generated a Vibrio alginolyticus mutant strain (V. alginolyticus△vscC) with attenuated virulence by knocking out the vscC encoding gene, a core component of type III secretion system (T3SS), which led to starkly reduced apoptotic rates in hemocyte hosts compared to the V. alginolyticusWT control. In comparative proteomics, it was revealed that distinct immune responses arose upon encounter with V. alginolyticus strains of different virulence. Quite strikingly, the peroxisomal and apoptotic pathways are activated by V. alginolyticusWT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus△vscC infection. Results for functional studies further show that V. alginolyticusWT strain stimulated respiratory bursts to produce excess superoxide (O2•−) and hydrogen peroxide (H2O2) in oysters, which induced apoptosis regulated by p53 target protein (p53tp). Simultaneously, a drop in sGC content balanced off cGMP accumulation in hemocytes and repressed the occurrence of apoptosis to a certain extent during V. alginolyticus△vscC infection. We have thus provided the first direct evidence for a mechanistic link between virulence of Vibrio spp. and its immunomodulation effects on apoptosis in the oyster. Collectively, we conclude that adaptive responses in host defenses are partially determined by pathogen virulence, in order to safeguard efficiency and timeliness in bacterial clearance.

Antimicrobial peptides are a fundamental component of mollusks’ defense systems, though they remain a thinly investigated subject. Here, infection by Vibrio parahemolyticus triggered a significant increase in antimicrobial activity in oyster plasma. By using PBS-challenged oysters as a control, plasma peptides from immunologically challenged oysters were subjected to peptidomic profiling and in silico data mining to identify bioactive peptides. Thirty-five identified plasma peptides were up-regulated post infection, among which, six up-regulated peptides (URPs) showed a relatively high positive charge. URP20 was validated with significant antibacterial activity. Virtually, URP20 triggered aggregation of bacterial cells, accompanied by their membrane permeabilization. Interestingly, URP20 was found to be active against Gram-positive and Gram-negative foodborne pathogens as well as Candida albicans, with no cytotoxicity to mammalian cells and mice. Our study provides the first large-scale plasma peptidomic dataset that identifies novel bioactive peptides in marine mollusks. Further exploration of peptide diversity in marine invertebrates should prove a fruitful pursuit for designing novel AMPs with broad applications.

phagosomes are task-force organelles of innate immune systems, and evolutionary diversity and continuity abound in the protein machinery executing this coordinately regulated process. in order to clarify molecular mechanisms underlying phagocytosis, we studied phagocyte response to beads and Vibrio species, using hemocytes of the Pacific oysters (Crassostrea gigas) as a marine invertebrate model. Phagosomes from different stages of phagocytosis were isolated by density-gradient centrifugation, and more than 400 phagosome-associated proteins were subsequently identified via high-throughput quantitative proteomics. In modeling key networks of phagosomal proteins, our results support the essential roles of several processes driving phagosome formation and maturation, including cytoskeleton remodeling and signal transduction by Rab proteins. Several endoplasmic reticulum (ER)-associated proteins were identified, while live cell imaging confirms an apparent intimate interaction between the ER and phagosomes. In further quantitative proteomic analysis, the signal transducers CgRhoGDi and CgPI4K were implicated. Through experimental validation, CgRhoGDi was shown to negatively regulate actin cytoskeleton remodeling in the formation of oyster phagosomes, while CgPI4K signaling drives phagosome maturation and bacterial killing. Our current work illustrates the diversity and dynamic interplay of phagosomal proteins, providing a framework for better understanding host-microbe interactions during phagosome activities in under-examined invertebrate species.

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Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+ 5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

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