Final Report Summary - INFBIOSYS (The role of Bacterial Multigene Families in Infection: Identification of Host Target Networks by High-throughput Protein Interactomics)
The genomes of pathogenic bacteria often contain multiple copies of genes with similar sequence, thus forming what is known as a multigene family. Multigene families arise by duplication of an ancestral gene and subsequent sequence diversification. Gene duplication is a flexible and efficient mechanism for expanding functionality and adapting to new niches. After a gene duplication event, one of the gene copies can still fulfil its original function whereas the other is free to evolve and diverge, with sequence variation affording functional innovation. Multigene families from bacterial pathogens generally encode proteins that are secreted or associated with the bacterial surface and thus likely to be involved in the host-pathogen interaction. They are also generally unique to a specific group of pathogens, suggesting an involvement in their distinctive pathogenic features. Moreover, within a bacterial genus, different multigene family members are found in genotypes or species differing in virulence and pathogenic properties.
In this project, we took a systems biology-based approach to investigate two model multigene families from the facultative intracellular pathogens Listeria and Rhodococcus equi. Listeria is a major gram-positive foodborne pathogen that invades non-phagocytic cells and traverses several host barriers, causing severe invasive infections. Two internalin (Inl) multigene family members are responsible for the invasive phenotype of Listeria, but additional inl genes are present in the genomes of both pathogenic and commensal / non-pathogenic species of the genus. In the pathogenic actinomycete R. equi, members of the Vap multigene family, encoded in a virulence plasmid, are required for intracellular survival in macrophages. Recent data also suggest that the specific combination of vap genes carried by specific host-adapted plasmid types determines the host tropism of R. equi.
To address the role of the inl and vap families in virulence, we systematically identified host targets by interaction proteomics using high-throughput yeast two-hybrid (Y2H) screens with large host (mammalian) cDNA prey libraries. The screens revealed that the listerial internalins are extremely promiscuous in their potential interactions with the host, with many binding partners being identified for each tested member from the pathogenic species L. monocytogenes and L. ivanovii (n > 400). Inl proteins from non-pathogenic Listeria had much less host binding partners in the screens. Interaction network and pathway analyses revealed an enrichment in targets involved in vacuolar traffic, cytoskeletal regulation and ubiquitination.
Comparatively, the Vap proteins from R. equi had in general less interaction partners, with an enrichment of targets within inflammation and immune regulation pathways. Taken together, our data suggest that bacterial proteins establish contacts with multiple host proteins and that the high-throughput screens just probably uncover the 'tip of the iceberg' of a whole universe of potential interactions. A new revolutionary view thus emerges in which microbial infection pathogenesis not only involves a limited set of decisive contacts between a few specific bacterial and host proteins, but also myriad of subtle interactions of potential significance in balancing the host-pathogen interplay.
Our protein-protein interaction studies are the first step towards gaining an understanding into the global role of the inl and vap proteins in infection. The protein-protein interaction data are currently guiding mechanistic cell biological research to confirm the significance of the observed interactions and to dissect the role in pathogenesis of specific Inl and Vap interactions with host proteins. Microbial surface-associated and excreted proteins like the Inl and Vap family members are at the forefront of host-pathogen exchanges and our research may lead to the identification of novel anti-infective targets.
In this project, we took a systems biology-based approach to investigate two model multigene families from the facultative intracellular pathogens Listeria and Rhodococcus equi. Listeria is a major gram-positive foodborne pathogen that invades non-phagocytic cells and traverses several host barriers, causing severe invasive infections. Two internalin (Inl) multigene family members are responsible for the invasive phenotype of Listeria, but additional inl genes are present in the genomes of both pathogenic and commensal / non-pathogenic species of the genus. In the pathogenic actinomycete R. equi, members of the Vap multigene family, encoded in a virulence plasmid, are required for intracellular survival in macrophages. Recent data also suggest that the specific combination of vap genes carried by specific host-adapted plasmid types determines the host tropism of R. equi.
To address the role of the inl and vap families in virulence, we systematically identified host targets by interaction proteomics using high-throughput yeast two-hybrid (Y2H) screens with large host (mammalian) cDNA prey libraries. The screens revealed that the listerial internalins are extremely promiscuous in their potential interactions with the host, with many binding partners being identified for each tested member from the pathogenic species L. monocytogenes and L. ivanovii (n > 400). Inl proteins from non-pathogenic Listeria had much less host binding partners in the screens. Interaction network and pathway analyses revealed an enrichment in targets involved in vacuolar traffic, cytoskeletal regulation and ubiquitination.
Comparatively, the Vap proteins from R. equi had in general less interaction partners, with an enrichment of targets within inflammation and immune regulation pathways. Taken together, our data suggest that bacterial proteins establish contacts with multiple host proteins and that the high-throughput screens just probably uncover the 'tip of the iceberg' of a whole universe of potential interactions. A new revolutionary view thus emerges in which microbial infection pathogenesis not only involves a limited set of decisive contacts between a few specific bacterial and host proteins, but also myriad of subtle interactions of potential significance in balancing the host-pathogen interplay.
Our protein-protein interaction studies are the first step towards gaining an understanding into the global role of the inl and vap proteins in infection. The protein-protein interaction data are currently guiding mechanistic cell biological research to confirm the significance of the observed interactions and to dissect the role in pathogenesis of specific Inl and Vap interactions with host proteins. Microbial surface-associated and excreted proteins like the Inl and Vap family members are at the forefront of host-pathogen exchanges and our research may lead to the identification of novel anti-infective targets.