The project seeks to understand the mechanisms underlying microbial symbiosis in the bee gut microbiome. In our first objective, we are using comparative genomics and transcriptomics to uncover factors that mediate inter-bacterial interactions. In particular, we have performed transcriptomics of the various members of the bee gut microbiota under conditions of in-vivo colonization of the bee host to determine which genes are turned on in the presence of the host, and in mono-inoculation as well as under co-colonization of pairs of strains. We found that different bacterial combinations elicit different responses, and are in the process of analyzing this data to find candidate genes which may be responsible for inter-bacterial interactions in the context of host colonization. Additionally, we have develped a computational pipeline to search the core members of the bee gut microbiota for various types of bacterial secretion systems. Secretion systems are molecular appratuses that enable bacteria to interact with and modify their neighbors and their environment. We have quantified the diversity of these systems in the bee gut symbionts, and have also used phylogenetics to reveal the evolution of these systems. We are currently in the final stages of this study, and are preparing a manuscript from the bioinformatic analyses results. We are also extending the analyses of secretion systems and other inter-cellular interaction mechanisms to gut metagenomes that are now available in online databases. We are identifying correlations between the prevalence and diversity of secretion systems to microbiome metadata such as host species, relatedness (bee colony), and geographic location. This will provide a first look into how host ecological factors play a role in the molecular ecology of genes encoding factors that mediate inter-cellular interactions. We have also have a longitudinal study, monitoring changes to microbiome composition and functional gene complement using metagenomic sequencing.
In our second objective, we are characterizing the proteins that are secreted by these secretion systems, particularly the type 6 secretion system (T6SS), which is known for mediating inter-cellular competition. We have cloned the C-terminal toxin domains from 32 effectors from the RHS family, the majority of which were found to have anti-bacterial properties when heterologously expressed in E. coli. These proteins have also been His-tagged, and will be purified to study biochemically. They will also be tested in yeast to test if they have anti-eukaryotic activity. Futhermore, bioinformatic work has been done to characterize the entire RHS complement of the bee gut microbiota, by searching for conserved RHS domains across sequenced whole genomes as well as metagenomes. We are using both conserved domain searches as well as 3D structure prediction (Alphafold) to predict their mode of action. Finally, we have created knockout mutants of T6SSs in key bee gut symbionts, and will use them in proteomic screens to identify which effectors are secreted by which secretion systems, as well as identify potential unknown effectors. Another mechanism of intercellular interaction we are investigating is the production of outer-membrane vesicles (OMV). OMVs can move proteins and nucleic acids between cells, thus modulating their transcriptional responses. We found that the bee gut symbionts are prolific producers of OMVs, and we are now characterizing their content and function using genomics and proteomics.
Finally, we are investigating how host and microbial transcription may be co-regulated in different gut compartments. To start, we are building a cell atlas of the bee hindgut, using single cell and spatial transcriptomics. This will lay the foundation for more mechanistic studies, ultimately revealing the cell types involved in host-microbe interaction, such as those with receptors of microbial ligands and those responsible for immune signalling processes.
