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

Abstract

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.

KPI5 Is Involved in the Regulation of the Expression of Antibacterial Peptide Genes and Hemolymph Melanization in the Silkworm, Bombyx mori

“…We further found that KPI5 binds to pathogens and their PAMPs but does not inhibit pathogen growth. Unlike KPI5, silkworm low molecular weight protease inhibitors SPINK7 (Kazal-type) and BmSPI51 (Kunitz-type) directly inhibit fungal growth by binding to β -d-glucan on the fungal surface ( 30 , 38 ). Insect immune processes mainly include: immune recognition, signal transmission, and effector production ( 48 ).…”

“…The binding between rKPI5 protein and PAMPs was measured using an enzyme-linked immunosorbent assay (ELISA) according to previous studies ( 37 , 38 ), with slightly modifications. Briefly, 5 μg LPS, PGN, and ZYM were dissolved in coating solution (0.5 M carbonate buffer, pH 9.8), added to a 96-well microtiter plate, and incubated at 4°C overnight.…”

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“…We further found that KPI5 binds to pathogens and their PAMPs but does not inhibit pathogen growth. Unlike KPI5, silkworm low molecular weight protease inhibitors SPINK7 (Kazal-type) and BmSPI51 (Kunitztype) directly inhibit fungal growth by binding to b-d-glucan on the fungal surface (30,38). Insect immune processes mainly include: immune recognition, signal transmission, and effector production (48).…”

“…The binding between rKPI5 protein and PAMPs was measured using an enzyme-linked immunosorbent assay (ELISA) according to previous studies (37,38), with slightly modifications. Briefly, 5 mg LPS, PGN, and ZYM were dissolved in coating solution (0.5 M carbonate buffer, pH 9.8), added to a 96-well microtiter plate, and incubated at 4°C overnight.…”

“…The in vitro encapsulation assay was performed as previously described (38,39), with slight modifications. Briefly, Ni-NTA agarose beads (Qiagen, Dusseldorf, Germany) were equilibrated in TBS (Tris-HCl pH 8.0, 100mM NaCl), and rKPI5 protein was added and incubated with the beads at 4°C overnight.…”