Kunitz-type protease inhibitor BmSPI51 plays an antifungal role in the silkworm cocoon

Zhang, Xiaolu; Guo, Kaiyu; Dong, Zhaoming; Chen, Zhiyong; Zhu, Hongtao; Zhang, Yan; Xia, Qingyou

doi:10.1016/j.ibmb.2019.103258

Cited by 26 publications

(25 citation statements)

References 22 publications

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“…At least seven protease inhibitor families with different domains were identified in the hemolymph, including serpin, TIL, Kazal, ITI, kunitz, WAP, and amfpi (4,6). Previous studies revealed that protease inhibitors with TIL and kunitz domains play antifungal roles in silkworms (23,24,27). Many studies have also found that serine protease inhibitors with Kazal domain (SPINKs) were also involved in animal immunity against bacterial and fungi (18,19,37).…”

Section: Discussionmentioning

confidence: 99%

“…Assay for inhibitory activity against protease was performed as previously described with modification (23)(24)(25)(26)(27). Trypsin, achymotyrpsin, pronase, subtilisin, thrombin were purchased from Sigma-Aldrich (St. Louis, USA), and proteinase K and papain were purchased from Sangon Biotech (Shanghai, China).…”

Section: Assay For Inhibitory Activity Against Proteasementioning

confidence: 99%

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SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections

Dong

et al. 2021

Front. Immunol.

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Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to β-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

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Section: Discussionmentioning

confidence: 99%

Section: Assay For Inhibitory Activity Against Proteasementioning

confidence: 99%

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections

Dong

et al. 2021

Front. Immunol.

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“…The abundance of protease inhibitors in wild silkworm cocoons is significantly higher than that in domestic silkworm cocoons, further confirming the role of protease inhibitors in resistance to microbial infection given that wild silkworms are exposed to more environmental stress than domestic silkworms [ 45 ]. Several types of protease inhibitors have been reported in silkworm cocoons, including Kunitz, Kazal, TIL, AMFPI, ITI, etc., many of which have been shown to play significant roles in insect defense functions [ 44 , 46 , 54 ]. These results suggest that sericin 1 and protease inhibitors cooperate in the fight against microbial infection.…”

Section: Discussionmentioning

confidence: 99%

Role of sericin 1 in the immune system of silkworms revealed by transcriptomic and proteomic analyses after gene knockout

Zhao

et al. 2021

FEBS Open Bio

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The domestic silkworm is a type of lepidopteran insect that feeds on mulberry leaves and has high economic value because of its ability to spin cocoons. Sericin 1 is an important component of silkworm cocoons, accounting for approximately 25% of the material. In this study, CRISPR/Cas9‐mediated gene editing was successfully used to destroy the sericin 1 gene, and homozygous mutants were obtained after continuous screening. Homozygous mutation resulted in premature termination of the translation of sericin 1 protein at 323 amino acids. Comparative transcriptomic and proteomic analyses of middle silk gland cells from wild‐type individuals and mutants were performed on the fourth day of the fifth instar, and the results suggest that sericin 1 plays an important role in the cellular immune system. In addition, the results suggest that sericin 1 has a synergistic effect with some protease inhibitors and that the secretion of these proteins is strictly regulated. These results will provide new insights into the function and expression pattern of sericin 1 and the mechanism of silk secretion.

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“…When P/N > 2.1, protein is considered to be bound to peptidoglycan [18][19][20]. For the peptidoglycan competition assay, 25 μg peptides was added to 50 μL 4 μg/μL PGN solution and 50 μL PBS solution as the experimental group and the control group, respectively, and incubated for 1 h. We added the incubated solution to a 96-well plate, and added an equal volume of PBS solution as a blank group.…”

Section: Peptidoglycan Binding and Competition Assaymentioning

confidence: 99%

Antibacterial Mechanism of Silkworm Seroins

Zhu

Zhang

et al. 2020

Polymers

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Seroin 1 and seroin 2 are abundant in silkworm cocoon silk and show strong antibacterial activities, and thus are thought to protect cocoon silk from damage by bacteria. In this study, we characterized the expression pattern of silkworm seroin 3, and found that seroin 3 is synthesized in the female ovary and secreted into egg to play its roles. After being infected, seroin 1, 2, and 3 were significantly up-regulated in the silkworm. We synthesized the full-length protein of seroin 1, 2, and 3 and their N/C-terminal domain (seroin-N/C), and compared the antimicrobial activities in vitro. All three seroins showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Seroin 2 showed better antibacterial effect than seroin 1 and 3, whereas seroin 1/2/3-N was better than seroin 1/2/3-C. We found that seroin 2-C has stronger peptidoglycan binding ability than seroin 2-N per the ELISA test. The binding sites of seroin 2 with bacteria were blocked by peptidoglycan, which resulted in the loss of the antibacterial activity of seroin 2. Collectively, these findings suggest that seroin 1 and 2 play antibacterial roles in cocoon silk, whereas seroin 3 functions in the eggs. The three silkworm seroins have the same antibacterial mechanism, that is, binding to bacterial peptidoglycan by the C-terminal domain and inhibiting bacterial growth by the N-terminal domain.

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Kunitz-type protease inhibitor BmSPI51 plays an antifungal role in the silkworm cocoon

Cited by 26 publications

References 22 publications

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections

Role of sericin 1 in the immune system of silkworms revealed by transcriptomic and proteomic analyses after gene knockout

Antibacterial Mechanism of Silkworm Seroins

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