Discerning the role of bus-1 in susceptibility of Caenorhabditis elegans to pathogenic Microbacterium nematophilum via an inducible protein degradation protocol using Auxin

Abstract

A host/pathogen interaction is dependent on many factors; this research focuses on susceptibility due to a host’s genome. In Caenorhabditis elegans, a Pax gene encodes for a transcription factor (EGL-38) that regulates genes expressed in the hindgut of the nematode. This hindgut function is important for mediating the response to the bacterial pathogen Microbacterium nematophilum. When a C. elegans becomes infected by M. nematophilum, the worm experiences severe constipation, a deformed anal region (DAR), and stunted growth. egl-38 mutants are resistant to M. nematophilum, whereas wild-type animals are sensitive. Gene expression microarray analysis of RNA from a wild type C. elegans verses egl-38 mutants identified many genes that might be important for mediating the sensitivity. We tested these candidates using a combination of RNAi knock down, genetic C. elegans mutant experiments and gene expression studies. These experiments identified one EGL-38 regulated gene, bus-1, that is required for M. nematophilum sensitivity. We also confirmed that bus-1 is specifically expressed in the hindgut. We have introduced BUS-1 protein (under control of a different promoter, egl-5) into an egl-38 mutant C. elegans and found that it restores sensitivity to the mutant. These findings further support that the bus-1 gene product is required for this host/pathogen interaction and that bus-1 is a key EGL-38 target gene. However, what is not clear is whether bus-1 plays a developmental role or a physiological role in mediating the host/pathogen response. In order to specifically target BUS-1, we are constructing an inducible protein degradation protocol using a plant hormone, Auxin. This system has been incorporated into yeast cultures and human cell lines and has the ability to induce the degradation of proteins tagged with a specific sequence within cells. We are generating plasmids to tag BUS-1 and observe the physiological response that occurs in the presence of M. nematophilum upon degradation of the bus-1 gene product. With the generation of these plasmids, we will be able to further analyze the effect that bus-1 has on this host/pathogen interaction.