Parasitic-like modulation of Bacillus thuringiensis development and larvicidal activity by a bacteriophage

Bacteria are preyed upon by bacteriophages (phages), which, like other viruses, hijack the host`s cellular machinery to replicate. It is becoming increasingly clear that phages and bacterial communities can have a significant impact on their eukaryotic host. In this project, we will investigate how the temperate phage GIL01, which infects the endospore-forming bacterium Bacillus thuringiensis serovar israelensis, favors its own and bacterial transmission to other trophic levels. It is worth noting that B. thuringiensis serovar israelensis is the most widely used natural, non-hazardous biopesticide against mosquito larvae in the world. To investigate phage-bacterial-larvae association, we will use the highly entomopathogenic B. thuringiensis serovar israelensis strain T0131 as the phage host, that was recently isolated from soil in Brazil. This isolate does not carry the GIL01 prophage (GIL01 genome) and we have produced a T0131 strain carrying the GIL01 prophage in the laboratory. In work package 1, we will demonstrate that the small 50-amino acid protein gp7, which is crucial for proper establishment of the lysogenic state of phage GIL01, or other determinants of phage GIL01 cause significant phenotipic changes in the host, strain T0131, e.g., we will use spore and plaque forming assays and transcriptomics to investigate the effects of GIL01 prophage determinants on enhancement of crystal toxin synthesis, entry into sporulation, endospore viability, and to show that GIL01 prophages package into endospores. This is important because endospores could allow safe entry of GIL01 into the insect body and thus may promote the transfer of GIL01 prophages entrapped in endospores to a higher trophic level, in larvae. It is worth noting that B. thuringiensis endospores have been shown to be ingested by insects and to germinate in the gastrointestinal tract. Our goal is also to show that the prophage GIL01, primarly via the gp7 protein is capable of rewiring the host transcriptomic profile, which enhances the pathogenesis of B. thuringiensis T0131 against insect larvae . Hence, we wish to postulate a concept that prophages can naturally enhance the bacterial biopesticide. We will test the effect of GIL01/gp7 on a major pest, spotted wing drosophila (Drosophila suzukii) and the lepidopteran model insect, grater wax moth (Galleria mellonellla), which are routinely used to develop innovative biopesticides. We will also outsource testing of larvicidal activity of the T0131 strain carrying the prophage GIL01 or not on Aedes aegypti larvae, the yellow fewer mosquito (BioGenious, Germany). To reconstruct the internal anatomy to assess gastric damage to midgut larvae, X-ray computerized tomography (micro-CT) of the larvae and scanning electron microscopy of the inner surface of the digestive tract will be performed. We will test the sporulation efficiency of strain T0131 carrying or not the prophage GIL01, when grown on larval cadavers. In work package 2, we will use confocal microscopy, quantitative PCR and proteomics to confirm that the phage GIL01 can enter the cytoplasm of insect cells. We also aim to show that gp7 and certain other determinants carried by the prophage GIL01 can be transcribed and synthesized in the insect cell, leading to extensive changes in the proteomic landscape of the insect cell. Thus, show that phage GIL01 can directly affect the insect. In work package 3 we aim to demonstrate that gp7-like proteins have also evolved and function as bacterial take-over factors in distant pathogenic Gram-negative bacteria. We will apply RNA-seq, surface plasmon resonance spectrometry, and crosslinking linked to mass spectrometry analysis to identify gp7-like proteins in the bacterial families Vibrionaceae and Enterobacteriaceae that have the potential to manipulate processes of members of these families that are important human pathogens. This will demonstrate that convergent evolution of gp7-like proteins has occurred in bacteria.