Main objectives/results:

SA1- identification of effector proteins

We have developed and applied computational pipelines to identify gut bacterial proteins that are likely translocated by via T3SS. We analyzed 161,115 proteins of human gut bacteria from strains available in culture collections, which resulted in identification of >1,800 predicted effectors, which were transferred to the interactome pipelines (below). In addition, 16,179 proteobacterial metagenome assembled genomes (MAGs) where screened for T3SS. The established workflow for identifying effectors was applied to 474,871 unique proteins, resulting in 2,891 putative effectors predicted by all methods. Quality ranking resulted in selection of 186 ‘metagenome effectors’, for which ORF reagents will be generated by gene synthesis.

SA2 - Microbiome-human interaction mapping

186 sequences were selected from metagenome samples and 1,810 sequences from 44 cultivated strains. For most effective cloning, 18 microbial strains with 1,307 predicted effectors were selected. Protocols for DNA isolation and PCR amplification needed to be developed and optimized for individual strains and application of optimized protocols resulted in 1,051 effector amplicons (>80% success). Cloning of these ORFs is in process. The human ORFeome and accessory reagents have been prepared for Y2H screening.

Interaction prediction: We have inferred ~500,000 interactions between 2,300 bacterial predicted effectors and the proteins of the human proteome. We are currently performing an quality assessment of the predictions using Montecarlo simulations. 

SA3 - relate human effector targets to functional and  disease modules

The European Nucleotide Archive (ENA) and PubMed were extensively searched to identify studies which offered WGS metagenomic data and meta-information about nutrition of the human individuals. 

In addition, 112 research articles were identified by collaborators which contain primary information about effector targets. Each article was reviewed by 2 persons and a comprehensive tabular overview containing effectors and their experimentally identified targets was created. The Positive Reference Set (PRS) for the mapping pipeline will be selected from these curated interactions. 

SA4 - Verification of human health

Results expected in later project state 

Expected project impact:

This project aims to shed light on functional and mechanistic questions basis of how microbes actively modulate the host as nutrition-mediated responses. An understanding of the mechanisms by which microbes can influence human molecular processes and ultimately human health will allow precise and informed health prevention and intervention strategies. 

Depending on the project results, informed nutritional intervention strategies can be developed to decrease disease risks. Disease modules overlapping with human effector targets can be alleviated by dietary or pharmaceutical interventions. 

Our results so far correspond to our project planning but are not yet at the stage where they can be communicated to the public as new insights.

To date our most important research results are the succesfull screening and effector predictions within 77 bacterial genomes  and constitution of a set of 1,800 predicted effectors. To our knowledge this result indicates for the first time the potential of bacterial proteins from non-pathogenic microbes in the microbiome that may directly influencing human intracellular processes. 

We analysed 161,115 proteins of human gut bacteria from strains available in culture collections for potential translocation via T3SS. In addition, 16,179 proteobacterial metagenome assembled genomes (MAGs) where screened for T3SS. The established workflow for identifying effectors was applied to 474,871 unique proteins, resulting in 2,891 putative effectors that were predicted by all methods. 186 sequences were selected from metagenome samples and 1,810 sequences from 44 cultivated strains. Experimentally 18 microbial strains containing 1,307 predicted effectors were selected and 1,051 pcr effector pcr fragments were obtained.