The trypsin inhibitor pro-peptide induces toxic effects in Indianmeal moth, Plodia interpunctella

https://doi.org/10.1016/j.pestbp.2020.104730Get rights and content

Highlights

  • Inhibitory effect of the pro-region against P. interpunctella trypsin was evaluated.

  • Five peptides were designed based on molecular docking simulations.

  • The peptide could successfully suppress the pest midgut trypsin in vitro.

  • The peptide did not show considerable inhibitory effects on mammalian trypsin.

  • Growth and development retardation were observed in pupa and adult insects.

Abstract

The inhibitory potential of an inhibitor peptide based on the pro-region of trypsin zymogen was investigated in Indianmeal moth, P. interpunctella, which is a world-wide insect pest of stored food. Five peptides were designed based on molecular docking simulations. The designed peptide with the best score was selected and synthesized for further screening in vitro and in vivo. The peptide was characterized and its inhibitory effects towards the insect trypsin were evaluated and the kinetic analysis revealed a competitive type of inhibition against the target enzyme. The results showed that the peptide could successfully suppress the pest midgut trypsin, and more interestingly, it did not show considerable inhibitory effects on a mammalian trypsin. We also aimed to assess the effect of dietary insect meal treated with different concentrations of the peptide and observed a significant growth and development retardation in pupa and adult insects fed with the inhibitor peptide. The outcomes of the present study suggest an efficient inhibitor peptide that could specifically bind the P. interpunctella trypsin and inhibit its activity, which would be safe against human being health and environment. Notably, this is the first report on in vivo assessment of the direct effect of a pro-region as the specific inhibitor in development as well as survival of the pest insect. Furthermore, our findings could be a promising for future designed pesticides used in pest management.

Introduction

Plants are the major source of food for human beings as well as animals that their productivity is influenced by pests (Hou et al., 2004). Among the pests, Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), is one of the most detrimental insect pests all around the world, which can cause severe damages to stored grain within a short time (Mohandass et al., 2007). Each year, this pest leads to a significant quantitative and qualitative loss of strategic stored products such as dried fruits, flour, nuts, and cereal grains (Jalali et al., 2015; Mohandass et al., 2007). For many years, traditional pesticides, due to their efficiency and economic benefit, were the first choice in control plan of the destructive pest (Cooper and Dobson, 2007). Unfortunately, despite the success in the control of food loss and waste, using conventional pesticides led to unfathomable challenges. Nowadays, their use has been limited mainly due to their high costs, negative effects on environmental pollution, potential damages to the human being, and the increasing levels of resistance to some pests (Benhalima et al., 2004; Isman, 2006). Therefore, there is an urgent need to develop an alternative safe pest control strategy with fewer harmful effects on human and environment (Kumar et al., 2014).

Plant defenses against insect herbivores are mediated, in part, by enzymes that impair digestive processes in the insect gut (Gatehouse and Gatehouse, 1998). In this regard, insect digestive enzymes are considered as targets for pest control and therefore, insecticidal proteins such as α-amylase and protease inhibitors could be a potential protective strategy against attacking insects (Jamal et al., 2013; Mehrabadi et al., 2011). Insects need essential amino acids through digestion of food proteins and accordingly, their digestive proteases catalyze the hydrolytic breakdown of peptide bonds in proteins to generate free amino acids necessary for growth and development. When the protease activity is blocked by using ingested protease inhibitors (PI), insect mortality increases by restricting the availability of essential amino acids. As a resistant mechanism to PIs, insects induce the upregulation of enzymes that degrade the PIs resulting in the induction of enzymes that resist inactivation by PIs, and also overproduction of the protease enzymes to maintain normal levels of gut proteolysis (Macedo and Freire, 2011; Shukle and Murdock, 1983).

Pro-regions of proteinase precursors play an important role in down-regulating activities of the enzymes. They are located at the N-terminus of the zymogen, an inactive precursor containing a signal peptide, a pro-region, and a mature protein (Baker et al., 1992; Jitonnom et al., 2012). They have two critical roles; they assist proper folding of the mature enzymes (Ikemura et al., 1987), and retain the enzyme as inactive form (Baker et al., 1993; Taylor et al., 1995). According to a previous report, the synthetic peptides, based on the pro-region of Manduca sexta (L.) midgut trypsin showed an inhibitory potency towards the mature enzyme (Taylor and Lee, 1997). In addition, the recombinant pro-region of papaya proteinase IV acted as an inhibitor of the digestive cysteine proteases of the insect pest, Colorado potato beetle (Visal et al., 1998). Jitonnom et al. also reported an inhibitor of Plutella xylostella (L.) midgut trypsin by modifications of the pro-region of the pest enzyme (Jitonnom et al., 2012).

In many Lepidopteran species, trypsin- and chymotrypsin-like serine proteases are among the main midgut digestive enzymes (Khan and James, 2008) and accordingly, they can be considered as attractive targets for pesticide design. Recent studies demonstrated that trypsin acts as the key digestive enzyme in the Indianmeal moth larval midgut, which is active in alkaline pH (Oppert et al., 1996; Zhu et al., 2000). A limited previous works indicated that the pro-peptide segment of the zymogen trypsin of insects have a potential to inhibit the enzyme activity, even though the inhibition mechanisms and molecular pathways involved in their insecticidal action have not been fully determined yet (Santamaria et al., 2015; Taylor and Lee, 1997; Visal et al., 1998). Accordingly, in this study, in silico and in vitro analysis as well as in vivo assessment of the pro-region inhibitor of P. interpunctella midgut trypsin have been performed. For this purpose, five pro-peptides with different sizes were rationally designed based on the pro-region of the insect trypsin and their binding affinity to the target enzyme was predicted in silico based on molecular docking simulations. Next, the best peptide was selected as the candidate for further in vitro and in vivo analysis. Then, the synthesized peptide was characterized and its inhibitory effects on the activity of P. interpunctella trypsin were evaluated. The outcomes of the present study suggest pro-peptides as a safe and efficient approach for development of new pesticides to control agricultural pests in future.

Section snippets

Chemicals

BAPNA (N-benzoyl-L-arg-ρ-nitroanilide), porcine trypsin, dimethyl sulfoxide (DMSO) and glycerol were purchased from Sigma (Saint Louis, MO, USA). Tris, CaCl2 and NaCl were provided from Bio Basic Inc. (Markham, Ontario, Canada). Trichloroacetic acid (TCA), trifluoroacetic acid (TFA), acetonitrile (ACN), casein and all other chemicals were reagent grade and obtained from Merck (Darmstadt, Germany). Diethylaminoethyl (DEAE) Sepharose resin was provided by Pharmacia (Sweden).

Sequence alignments

Multiple sequence

Multiple alignment and pro-peptides design

A multiple sequence alignment of the selected trypsinogens was performed (Fig. 1 a). Comparison demonstrated that the sequence identity was insignificant (29–30%) between the trypsinogens of mammalians (H. sapiens, B. taurus, Sus scrofa) and P. interpunctella (Fig. 1 b) confirming a low structural similarity between their structures (RMSD = 3.17–3.44). In addition, the amino acid sequence of the insect trypsin showed 62% and 35% identity with P. xylostella and F. oxysporum, respectively (Fig. 1

Discussion

The cosmopolitan Indianmeal moth, P. interpunctella, is a serious insect pest of stored products and it has become one of the most important pests of processed food in the world (Jalali et al., 2015b; Masoumzadeh et al., 2017). For many years, the control of P. interpunctella has traditionally involved the use of conventional insecticides, but the exclusive use of traditional pesticides is detrimental to human health and environments (Jamal et al., 2013; Lawrence and Koundal, 2002). Due to the

Acknowledgments

This work was supported by the research council of Tarbiat Modares University, Tehran, Iran and Ministry of Sciences, Researches, and Technology, Iran.

Declaration of Competing Interest

None

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