Preliminary investigation of the immune activity of PmH2A-derived antimicrobial peptides from the pearl oyster Pinctada fucata martensii.

Antimicrobial peptides (AMPs) play important roles in the immune defense against pathogenic microorganisms. For instance, histone 2A (H2A)-derived AMPs is an antimicrobial peptide involved in the host's innate immune defense and immunoregulation. AMPs have been isolated from the pearl oyster Pinctada fucata martensii but their role in host defense remains poorly understood. To elucidate the structural features of P. f. martensii H2A (PmH2A)-derived AMPs and their potential immune functions, we synthesized a series of laboratory-designed synthetic analogs of PmH2A and examined their antimicrobial properties, as well as their mechanisms of action. This analysis revealed inhibitory effects on the growth of Gram-positive and Gram-negative bacteria. Further assessment by transmission electron microscopy (TEM) of two of the three peptides, PmH2A-AMP and PmH2A-AMP(5-13)[KLLK]3, confirmed that it exerted an anti-bacterial activity through membrane lysis. Finally, we found that the hemocytes and gills of P. f. martensii released antimicrobial H2A histones in response to LPS exposure, mimicking tissue damage and infection. This immune response is reminiscent of the neutrophil extracellular traps (NETs) recently described in oysters. Thus, the LPS challenge is sufficient to induce histone-derived peptide accumulation in pearl oyster P.f. martensii.

Members of the histone-derived antimicrobial peptide family from the pearl oyster Pinctada fucata martensii: Inhibition of bacterial growth.

Because it is difficult to isolate standard antimicrobial peptides (AMPs) using traditional biochemical approaches, we designed, synthesized, and evaluated a series of structurally altered histone-derived AMPs (HDAPs) from the pearl oyster Pinctada fucata martensii using molecular cloning approaches. Four histone-homolog genes (PmH2A, PmH2B, PmH3, and PmH4-1) were identified, of which PmH2A and PmH2B had yet to be described. PmH2A and PmH2B were therefore cloned using Rapid Amplification of cDNA Ends (RACE) and characterized. Constitutive PmH2A and PmH2B mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the gonads. Because α-helical content, hydrophilicity index, and the presence of a proline hinge may be the three important factors influencing the antimicrobial efficacy of HDAPs, we synthesized a series of eight N- and C-terminally truncated or amino acid-substituted synthetic candidate HDAP analogs derived from PmH2A, PmH2B, PmH3, and PmH4-1. Only the PmH2A- and PmH4-derived AMPs inhibited bacterial growth. The PmH2A-derived AMPs were α-helical proteins, while the PmH4-derived AMPs were extended strand/random coil proteins. Our results suggested that having an α-helical structure was particularly important for the antibacterial efficacy of the PmH2A-derived peptides; amphipathic structures (hydrophilic index, 0.3 to -0.3) may enhance the antimicrobial function of both the PmH2A- and PmH4-derived peptides. The high antibacterial efficacy of one of the HDAP analogs studied, PmH2A-AMP (5-13) [KLLK]3, indicated that this protein may represent a promising candidate for the treatment of bacterial infections in aquaculture mollusk species. This first study of HDAPs from the pearl oyster P. f. martensii provides new insights into the design and function of highly effective antimicrobial peptides.

Ihog proteins contribute to integrin-mediated focal adhesions.

Integrin expression forms focal adhesions, but how this process is physiologically regulated is unclear. Ihog proteins are evolutionarily conserved, playing roles in Hedgehog signaling and serving as trans-homophilic adhesion molecules to mediate cell-cell interactions. Whether these proteins are also engaged in other cell adhesion processes remains unknown. Here, we report that Drosophila Ihog proteins function in the integrin-mediated adhesions. Removal of Ihog proteins causes blister and spheroidal muscle in wings and embryos, respectively. We demonstrate that Ihog proteins interact with integrin via the extracellular portion and that their removal perturbs integrin distribution. Finally, we show that Boc, a mammalian Ihog protein, rescues the embryonic defects caused by removing its Drosophila homologs. We thus propose that Ihog proteins contribute to integrin-mediated focal adhesions.

Gene cloning and functional study of PmKSPI from Pinctada fucata martensii.

Kunitz-type serine protease inhibitors (KSPI) are a family of serine protease inhibitors (SPIs) and are extensively found in animals, plants, and microbes. SPI can inhibit proteases that may be harmful or unwanted to its cells. Here, a four-domain Kunitz-type SPI, PmKSPI, was cloned by RACE in the pearl oyster Pinctada fucata martensii. The full-length cDNA sequence of PmKSPI was 1318 bp, including the 5' UTR (25 bp), the 3' UTR (96 bp) and ORF (1197 bp). Homology analysis indicated that PmKSPI had the highest resemblance (30.14%) with its homolog in Crassostrea gigas. Phylogenetic analysis revealed that PmKSPI clustered with homologs in other molluscs. We found that PmKSPI mRNA expression in P. f. martensii was distributed in all six tissues, with the highest level in the mantle, and almost no expression in other tissues. After PAMPs challenge, expression of PmKSPI mRNA in the mantle was significantly up-regulated. The recombinant protein rPmKSPI significantly inhibited the growth of 5 kinds of Gram-negative bacteria but had little effect on Gram-positive bacterial activity. Transmission electron microscopy showed that plasmolysis occurred in two Gram-negative bacteria species when treated with rPmKSPI. rPmKSPI may thus have a bactericidal effect by destroying the bacterial cell membrane or cell walls and releasing its contents. Therefore, our results suggest that PmKSPI is tightly associated with the immunological defence of P. f. martensii.

Cloning and functional analysis of a trypsin-like serine protease from Pinctada fucata martensii.

Trypsin-like serine proteases (TLSs) play various roles in dietary protein digestion, hemolymph coagulation, antimicrobial peptide synthesis, and, in particular, the rapid immune pathways activated in response to pathogen detection. The cultured pearl industry, of which Pinctada fucata martensii is one of the most important species, is plagued by disease, thus leading to large economic losses. Herein, the molecular mechanisms underlying the innate immune response of P.f. martensii were explored. First, immune effector molecules from the P.f. martensii genome were screened and a TLS-like gene encoding a protein with a trypsin domain, herein designated as PmTLS, was identified. A multi-sequence alignment indicated a low sequence homology between PmTLS and other mollusk TLS-like proteins. Furthermore, a neighbor-joining phylogenetic analysis indicated that PmTLS has the closest genetic relationship to a Crassostrea gigas TLS. Additionally, real-time quantitative PCR (qPCR) analysis showed that PmTLS mRNA is constitutively expressed in all of the 6 examined P.f. martensii tissues, with significantly higher expression noted in hemocytes relative to the other tissues examined (p < 0.05). P.f. martensii samples were then challenged with various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide, peptidoglycan, and polyinosinic acid. In the challenge groups, PmTLS was significantly upregulated in hemocytes at 48 h post-challenge when compared to the unchallenged controls. Furthermore, treatment with recombinant PmTLS (rPmTLS) also significantly inhibited the growth of most of the examined gram-negative bacteria tested in vitro (p < 0.05), but it had little effect on the growth of the examined gram-positive bacteria. When examining morphological changes via transmission electron microscopy, rPmTLS treated bacteria exhibited morphological changes such as plasma wall separation. Thus, rPmTLS appears to play a bactericidal role by destroying bacterial cell membranes or cell walls, which subsequently leads to a release of the cellular contents and cell death. The findings presented herein have enabled further characterization of the immune defense mechanisms in P.f. martensii and may lead to improved disease control methods for the pearl cultivation industry.

Comparative proteomics and transcriptomics illustrate the allograft-induced stress response in the pearl oyster (Pinctada fucata martensii).

Implantation of a spherical nucleus into a recipient oyster is a critical step in artificial pearl production. However, the molecular mechanisms underlying the response of the pearl oyster to this operation are poorly understood. In this research, we used transcriptomic and proteomic analyses to examine allograft-induced changes in gene/protein expression patterns in Pinctada fucata martensii 12 h after nucleus implantation. Transcriptome analysis identified 688 differential expression genes (DEGs) (FDR<0.01 and |fold change) ＞ 2). Using a 1.2-fold increase or decrease in protein expression as a benchmark for differentially expressed proteins (DEPs), 108 DEPs were reliably quantified, including 71 up-regulated proteins (DUPs) and 37 down-regulated proteins (DDPs). Further analysis revealed that the GO terms, including "cellular process", "biological regulation" and "metabolic process" were considerably enriched. In addition, the transcriptomics analysis showed that "Neuroactive ligand-receptor interaction", "NF-kappa B signaling pathway", "MAPK signaling pathway", "PI3K-Akt signaling pathway', "Toll-like receptor signaling pathway", and "Notch signaling pathway" were significantly enriched in DEGs. The proteomics analysis showed that "ECM-receptor interaction", "Human papillomavirus infection", and "PI3K-Akt signaling pathway" were significantly enriched in DEPs. The results indicate that these functions could play an important role in response to pear oyster stress at nucleus implantation. To assess the potential relevance of quantitative information between mRNA and proteins, using Ward's hierarchical clustering analysis clustered the protein/gene expression patterns across the experimental and control samples into six groups. To investigate the biological processes associated with the protein in each cluster, we identified the significantly enriched GO terms and KEGG pathways in the proteins in each cluster. Gene set enrichment analysis (GSEA) was used to reveal the potential protein or transcription pathways associated with the response to nuclear implantation. Thus, the study of P. f. martensii is essential to enhance our understanding of the molecular mechanisms involved in pearl biosynthesis and the biology of bivalve molluscs.

Evidence for anisotropic spin-triplet Andreev reflection at the 2D van der Waals ferromagnet/superconductor interface.

Fundamental symmetry breaking and relativistic spin-orbit coupling give rise to fascinating phenomena in quantum materials. Of particular interest are the interfaces between ferromagnets and common s-wave superconductors, where the emergent spin-orbit fields support elusive spin-triplet superconductivity, crucial for superconducting spintronics and topologically-protected Majorana bound states. Here, we report the observation of large magnetoresistances at the interface between a quasi-two-dimensional van der Waals ferromagnet Fe0.29TaS2 and a conventional s-wave superconductor NbN, which provides the possible experimental evidence for the spin-triplet Andreev reflection and induced spin-triplet superconductivity at ferromagnet/superconductor interface arising from Rashba spin-orbit coupling. The temperature, voltage, and interfacial barrier dependences of the magnetoresistance further support the induced spin-triplet superconductivity and spin-triplet Andreev reflection. This discovery, together with the impressive advances in two-dimensional van der Waals ferromagnets, opens an important opportunity to design and probe superconducting interfaces with exotic properties.

Antimicrobial properties and immune-related gene expression of a C-type lectin isolated from Pinctada fucata martensii.

C-type lectins are carbohydrate-binding proteins that play important roles in the innate immune response to pathogen infections. Here, multi-step high performance liquid chromatography (HPLC), combined with mass spectrometry (MS), was used to isolate and identify proteins with antibacterial activity from the serum of Pinctada fucata martensii. Using this method, we obtained a novel isoform of C-type lectin (PmCTL-1). PmCTL-1 strongly inhibited gram-positive bacteria. The complete cDNA sequence of PmCTL-1 was 636 bp in length, and encoded a protein 149 amino acids long, containing a typical carbohydrate recognition domain (CRD). A phylogenetic analysis based on a multiple sequence alignment indicated that PmCTL-1 was highly similar to C-type lectins from other mollusks. Fluorescent quantitative real-time PCR analysis showed that PmCTL-1 mRNA was strongly upregulated in the mantle of healthy P.f. martensii, but was expressed only at low levels in the gill, gonad, hepatopancreas, adductor muscle, and hemocytes. PmCTL-1 expression levels in the mantle and hemocytes increased significantly in response to bacterial stimulation. This study provides a valuable framework for further explorations of innate immunity and the immune response in mollusks.

Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway.

The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. Here we performed a genetic screen in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and identified the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, which are involved in the early secretory pathway of Wingless/Wnt. We provided genetic evidence demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. We found that Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, we showed that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, our data provide important insights into the early secretory pathway of Wg/Wnt.