Breaking the walls to wake-up bacterial cells

Bacteria can survive harsh environmental conditions by slowing down their major living processes. They look like normal cells but behave like the dead ones until stimulated by proteins called the Resuscitation-promoting factors (Rpfs). Rpfs are constantly sectereted by normal growing bacteria and play an important role in making of cell envelope. However, sleeping or dormant bacteria do not produce the Rpfs. The main goal of this project is to find out how Rpfs wake-up sleeping cells. Rpfs are enzymes which digest bacterial cell wall (like lyzosyme) but unlike the latter they don't kill cells and somehow modify their envelope. Rpfs may simply break quite inflexible cell wall of sleeping cells and make them 'free' to grow and expand. In more sophisticated model fragments of cell wall, released by Rpfs during digestion, have a very special function as signalling molecules. In other words they are waking-up messages for dormant cells. These 'signals' bind to other molecules on the bacterial surface and initiate a complex system called a protein kinase cascade, a well described mechanisms of cell regulation in multicellular organisms. To address both hypotheses the investigator will isolate the major part of bacterial envelope, called murein and digest it with different Rpfs. The resulting products will be used for resuscitation of dormant cells and their composition will be analysed and characterised. Other experiments will be designed to establish what bonds the Rpfs cut in murein and how this cleavage influences the structure of bacterial cell envelope. In separate part of the project the investigators will attempt to understand how bacterial cells control the enzymes which able to destroy them by 'eating' bacterial envelope. Rpfs proteins will be applied as an example of such cell-wall degrading enzymes. The results of the project will not only satisfy scientific curiosity on bacterial cell function but also provide novel methods for manipulating of physiological condition of bacteria and further improving their application in biotechnology, medicine and specifically for combating of persisting infections caused by dormant bacteria.

Rpfs are a family of proteins secreted by actively growing cells. They resuscitate non-replicating bacteria and stimulate growth of cells in nutrient poor and stressful conditions. Rpfs are cell-wall hydrolysing enzymes and their enzymatic activity is essential for the biological effects. However, the precise mechanisms of Rpf-mediated resuscitation as well as the specificity of Rpf enzymes and released products remain unknown. The investigators will address two current hypotheses explaining the phenomenon of Rpf-mediated resuscitation of mycobacteria (i) Rpfs directly modify murein specific to dormant cells and enables growth activation; (ii) Rpf-derived muropeptides serve as signalling molecules that stimulate bacterial resuscitation. The proposed experiments will fill the existing gap in the Rpf-related research by identifying the bonds cleaved by Rpfs in murein and characterising structure of Rpf-derived muropeptides. Alternate sources of murein will be used for these experiments, including E. coli murein which is naturally susceptible to Rpf digestion, surrogate murein produced in genetically modified E. coli and mycobacterial peptidoglycan. The results will provide a basis for production of Rpf-derived muropeptides required for structural and physiological studies. It is likely that Rpf activity is modulated by other proteins. These will be searched for in a novel two plasmid screen for extracellular protein interactions based on the ability of Rpfs to induce E. coli lysis in vivo. The findings have the potential to enable controlled production of Rpf-dependent cells and other Rpf-related reagents. Delivery of the proposed project will result in better understanding of fundamental biological processes and improvement of Rpf application in industry and medicine.

The proposed research will impact on basic science by uncovering the mechanisms of bacterial survival and resuscitation of dormant cells or 'revival of immortals'. Apart of educational value this knowledge will provide a basis for scientific experiments and design of novel methods. The PI is aware of difficulties in production of Rpf-dependent cells, important model organisms for study of non-replicating persistence, and determined to explore new methodologies for establishment of physiological assays for Rpf proteins. Development of reliable procedure for production of muropeptides and their application in structural biology is another important result emerging from this project. The proposed programme will also impact on applied sciences by offering novel opportunities for drug design, development of diagnostic tools and means for improvement of industrially important microorganisms. It is generally accepted that non-replicating bacteria are tolerant for antibiotics; therefore knowledge on reactivation of such microorganisms will promote formulation of novel strategies for chemotherapy. The wider translational potential includes detection of pathogenic bacteria in environment), control of disease transmission and reactivation. Altogether these useful applications will impact on human well-being and promote scientific progress.