Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera.

Visweshwar Regode,Hari C Sharma,Sreeramulu Kuruba,Akbar S Mohammad

doi:10.3389/fmicb.2016.01567

Abstract

Bacillus thuringiensis toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli (CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of B. thuringiensis protoxin and play a major role in inducing pathogenicity of B. thuringiensis toxins in H. armigera.

Highlights

Cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is polyphagous pest
After ingestion by the larvae, the B. thuringiensis protoxin undergo proteolysis in midgut under the alkaline environment, the active toxin formed will binds to receptors on the midgut epithelium, and active toxin inserted into the membrane, creates membrane pores and eventually results in cell lysis leading to death of insect (Grochulski et al, 1995; Aronson and Shai, 2001)
In earlier studies (Visweshwar et al, 2015), we found that the insecticidal activity of B. thuringiensis toxins was reduced in H. armigera larvae eliminated with gut bacteria using antibiotic cocktail

Summary

Introduction

Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is polyphagous pest. Plants expressing toxin genes produced from the bacteria Bacillus thuringiensis have been deployed on a large scale for controlling insect pests (Sharma et al, 2004; James, 2013). Pest insects had gained resistance to B. thuringiensis toxins and the cause of resistance may be due to lack of major gut protease involved in protoxin cleavage (Oppert et al, 1997), variations in the expression pattern of midgut proteases (Keller et al, 1996; Karumbaiah et al, 2007), improper processing of protoxin by proteases (Li et al, 2004; Rajagopal et al, 2009), and reduced binding of the active toxin to the receptors on midgut epithelium (Wang et al, 2007; Nair et al, 2013). Resistance to B. thuringiensis toxins is associated with changes in expression of receptors, glycosylphosphatidyl-inositol (GPI) anchored alkaline phosphatases (ALPs) (Fuentes et al, 2011), GPI anchored aminopeptidases-N (APN) (Tiewsiri and Wang, 2011), cadherin (CAD) (Fabrick et al, 2014), and ABC transporter loci (Baxter et al, 2011)

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