In Silico and Molecular Analysis of Xpta Protein Toxin Genes of Xenorhabdus Sp. And Efficacy of the Bacterium on Sitophilus Zeamais and Prostephanus Truncatus.

Abstract

Bacteria of the genus Xenorhabdus are entomopathogens that produce insecticidal protein toxins against a wide range of insects. The main proteins involved are the Xenorhabdus protein toxins (Xpts), categorized as class A, B and C. They work best as a complex, though individually, XptA has been found to be more potent against some insect pests such as Pieris brassicae and Heliothis virescens. The maize weevil (Sitophilus zeamais) and the larger grain borer (Prostephanus truncatus) are storage insect pests that cause the greatest postharvest losses of maize in Kenya, generally estimated to range between 20-30% annually. Many of the control strategies used against these pests have significant drawbacks. In particular, chemical insecticides are costly and may pose health and environmental hazards. There is thus a need to find an efficient and sustainable alternative. This project sought to identify an isolate of Xenorhabdus sp. that is effective against the two storage pests, Sitophilus zeamais and Prostephanus truncatus. It also sought to isolate and analyse the Xpt proteins that are involved in insecticidal activity. The bacterium was characterized using morphological features typical to the genus, as well as, molecularly via a 16S rDNA sequence homology search. Bacterial cells were lysed to isolate the proteins and the crude lysate purified using a size exclusion column before separation on a PAGE gel. The target bands were identified by comparison to a protein ladder. The insecticidal activity of Xenorhabdus sp. against the target pests was then tested by incorporation of live bacteria into an artificial diet before feeding the insects with it. XptA sequences as well as those of their homologs were then retrieved from databases and analysed using bioinformatics techniques and tools. The bacterium was identified as Xenorhabdus griffinae. Purified protein bands were found in the expected size range indicating that they were XptA. Whole bacterial cells caused mortality of S. zeamais )05.0;32.78(22p and reduced consumption of maize pellets by P. truncatus )05.0;87.117(24p . Through multiple sequence alignment, XptA, TcdA and SepA genes were found to be homologous. Similarly, through superimposition of protein tertiary structures on MATRAS and Pymol, XptA, TcdA and SepA were found to be closely related, thus indicating a common mode of action. XptA were found to be homologous to TcdA2, SepA and TccA proteins that have been reported to show toxicity to mammals and plants. All the proteins analysed were found to contain similar conserved domains among which was VRP1, a domain originally found in the virulence plasmid protein SpvA of Salmonella spp.. Seven motifs were identified in the VRP1 domain. Of these, motifs number three and four, as well as their inter-motif sequences, were highly conserved across the board while the other motifs were absent in the XptA, TcdA and SepA sequences. A homology search on the HHpred server using both the VRP1 and full class A amino acid sequences, returned TcdA1 (PDB ID: 1VW1A) Tc toxin crystal structure from P. luminescens as the PDB top hit. The Xenorhabdus sp. used in this study showed potential for use in control of these storage insect pests. Furthermore, through bioinformatic analysis, the XptA proteins also showed a potential for use in pest control. Therefore, overall, Xenorhabdus sp. and their class A proteins, could be useful in the development of a cleaner, greener and sustainable crop pest control strategy.