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

Abstract

Kunitz-type protease inhibitors (KPIs) are ubiquitously found in many organisms, and participate in various physiological processes. However, their function in insects remains to be elucidated. In the present study, we characterized and functionally analyzed silkworm KPI5. Sequence analysis showed that KPI5 contains 85 amino acids with six conserved cysteine residues, and the P1 site is a phenylalanine residue. Inhibitory activity and stability analyses indicated that recombinant KPI5 protein significantly inhibited the activity of chymotrypsin and was highly tolerant to temperature and pH. The spatio-temporal expression profile analysis showed that KPI5 was synthesized in the fat body and secreted into the hemolymph. In vivo induction analysis showed that the expression of KPI5 in the fat body was significantly upregulated by pathogen-associated molecular patterns (PAMPs). Binding assays suggested that KPI5 can bind to pathogens and PAMPs. In vitro pathogen growth inhibition assay and encapsulation analysis indicated that KPI5 can neither kill pathogenic bacteria directly nor promote the encapsulation of agarose beads by silkworm hemocytes. Recombinant protein injection test and CRISPR/Cas9-mediated knockdown showed that KPI5 promotes the expression of antimicrobial peptides (AMPs) in the fat body. Moreover, the survival rate of individuals in the KPI5 knockdown group was significantly lower than that of the control group after pathogen infection. Phenoloxidase (PO) activity assays showed that KPI5 significantly inhibited the hemolymph PO activity and melanization induced by PAMPs. These findings suggested that KPI5 plays a dual regulatory role in innate immunity by promoting the expression of antimicrobial peptides in the fat body and inhibiting hemolymph melanization. Our study furthers the understanding of the function of insect KPIs and provides new insights into the regulatory mechanism of insect immune homeostasis.

The immune response mechanism of Nilaparvata lugens against a combined infection of rice ragged stunt virus and Metarhizium anisopliae

“…In insect innate immunity, serine protease cascade plays a vital role by regulating AMP production, PPO activation, and melanization cascade to fight against alien pathogen invasion and sustain insect survival. 17,21,23,45,46 In this study, a clip domain serine protease, named Venomase (NlVenomase), and a Kunitz-type serine protease inhibitor (NlKPI) were cloned in BPHs (Fig. S1).…”

“…[12][13][14][15] More importantly, a wide variety of harmful byproducts derived from immune reactions could also damage the host's physiological systems and even cause insects to die. 16,17 Melanization's main immune effect is to produce melanin to remove pathogens, which requires the proteolytic activation of prophenoloxidase (proPO) to its active form. 6,16 During this process, byproducts, such as quinone-like substances, reactive oxygen (ROI), and other harmful matter, damages host tissues and cells.…”

“…6,[18][19][20] Consequently, the intensity and duration of insect immune responses are strictly and precisely regulated to balance the relationships among immune effects synthesis, the generation of harmful byproducts, and energy consumption. 6,17 The serine protease cascade is an essential pathway in insect humoral immunity that regulates AMP synthesis, polyphenol oxidase (PPO) activation, and melanization reaction. 6,19,21 After successfully penetrating physical barriers, invading pathogens are recognized by pathogenic-related receptors (PRRs) of insect hosts, leading to the amplification of the serine protease cascade.…”

“…The serine protease cascade and other immune pathways were negatively regulated by the serine protease inhibitor (serpin) to avoid the excessive accumulation of harmful byproducts. 24,25,28 Heng et al 17 demonstrated that one of the Kunitz-type protease inhibitors (KPIs), which belongs to the family of serine protease inhibitors, plays a dual regulatory role in the Bombyx mori immune response. KPI5 promotes the expression of AMPs in the fat body and inhibits hemolymph melanization.…”

“…Compared with other insects, knowledge about the serine protease cascade in BPHs is extremely limited. 17,26 In the present study, we identified and cloned two genes in the serine protease cascade, designated to NlVenomase and NlKPI, to explore their role in an immune response. NlVenomase is a clip-domain serine protease and NlKPI is a Kunitz-type serine protease inhibitor.…”