Research project P7/28 (Research action P7)
Many of the microbial processes important for the establishment of infections by bacteria or fungi can be considered “developmental processes". For example, the transition from a planktonic cell to a biofilm cell attached to the host mucosal surface requires an intricate network of regulatory processes that determine the expression levels of a large number of genes. Similarly, the differentiation of microbial cells with normal physiology into antimicrobial-tolerant persister cells is another example of a developmental process involving strict regulation of metabolism and gene expression. Lastly, the successful colonization and development of infection in a human host requires the spatio-temporal regulation of the expression of virulence genes. The tight regulation of gene expression required to successfully complete these developmental processes is often directly influenced by complex interactions between microbial cells.
The overall aim of this project is to elucidate the regulatory mechanisms involved in key developmental processes in microorganisms. To achieve this goal we have assembled a consortium of seven Belgian research teams and one international team. These eight partners will jointly carry out tasks detailed in six work packages (WPs) :
WP1 : Tools
WP2 : Role of nutrient sensing in the development of single and multispecies biofilms
WP3 : Persistence
WP4 : The role of sRNA and proteins interacting with sRNA in microbial developmental processes
WP5 : The role of intercellular communication in microbial developmental processes
WP6 : Differentiation of and infection by -Proteobacteria
Sophisticated tools for detailed molecular analysis of microbial cells have fuelled novel discoveries in microbiology. Throughout this proposal, novel tools will also take a prominent place in the analysis of microbial differentiation, virulence, and biofilms in in vitro and in vivo situations. In WP1 a number of unique state-of-the-art tools will be (further) developed, and will be implemented and/or optimized. These tools will be put at the disposal of all partners.
The main goal of WP2 is to gain a better understanding of the role of nutrient sensing in the development of single and multispecies biofilms in different conditions. For this we will study single species bacterial (Staphylococcus aureus) and fungal (Candida albicans) biofilm formation as well as multispecies biofilm formation (C. albicans-S. aureus). The investigation of the molecular processes involved in the development and maintenance of these biofilms will be done by high-throughput transcriptomics (RNAseq).
To obtain a detailed view of the mechanisms underlying the development of highly tolerant persister cells, we will follow two approaches in WP3. In the first one, we will analyze the molecular mechanisms underlying the regulation of known genes involved, as well as the molecular activity of their products, including toxin-antitoxin systems. The second approach will be exploratory and will allow the identification of additional functions involved in persistence.
The hypothesis that sRNA play an essential role in the rewiring of the expression of genes involved in developmental processes, including biofilm formation, in Gram-negative bacteria will be investigated in WP4. A first goal in this WP is to identify and characterise sRNA genes involved in biofilm formation and maintenance in Burkholderia cenocepacia, using RNAseq. Subsequently, the specific role(s) of these sRNA in development of single- and multispecies biofilms will be elucidated through the characterisation of deletion and overexpression mutants. A second goal is the detailed characterisation of the role of CsrA, a protein known to interact with sRNA, in Escherichia coli.
In WP5 we will address a range of basic questions related to quorum sensing and quorum quenching mechanisms in Gram-positive and Gram-negative bacteria. We will investigate the QS mechanisms affecting the development of virulence in Brucella-infected cells and investigate at which stage of its vacuolar trafficking these are important. Futher we will elucidate how sRNA affect the regulation of quorum sensing in Burkholderia spp. And how Rgg/ComR-like QS regulatory systems affect population dynamics of various streptococci.
WP6 is devoted to the study of three aspects of the bacterial cell cycle that are typical for differentiated cell types in the -Proteobacteria Rhizobium etli and Brucella abortus. These three aspects are growth sites, initiation of genome replication and cellular asymmetry. For each aspect, we will use molecular markers that could detect these events in bacteria interacting with their host. In addition, we will characterize the differentiated cell types at the molecular level, using transcriptomics and metabolic analysis. Finally, we propose to investigate the molecular mechanisms regulating the transition between differentiated cells types.
In summary, we are confident that this integrated network will allow us to go beyond the state of the art and that our joint research will lead to groundbreaking findings in the field of microbial developmental processes.
