Protein import through the E. coli cell envelope

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We are rapidly running out of antibiotics for use in medicine and agriculture due to the inexorable rise of antibiotic resistant bacteria. The failure of drug discovery programmes in the pharmaceutical industry to furnish any new classes of antibiotics in over 30 years means our society is rapidly approaching a time akin to the pre-antibiotic era where routine operations carried a high degree of risk from potentially lethal infections, a view emphasized by recent reports on antimicrobial resistance from across the globe. Moreover, protecting our food against bacterial pathogens is an increasing concern; for example, contamination of food products by E. coli O157:H7 in 2011 across Europe resulted in multiple deaths.Clearly, new approaches are needed to respond to this problem. A group of molecules that have yet to be exploited in the treatment of bacterial infections are protein bacteriocins. Bacteriocins are protein antibiotics produced by bacteria to kill their close related neighbours during competition for nutrients. This proposal focuses on one class of bacteriocins known as colicins. Colicins are made by the bacterium Escherichia coli to kill other E. coli, and have recently been shown to be highly effective at controlling E. coli O157:H7 infections in food crops.My proposal is focused on understanding how these toxic proteins, which have no activity against human cells, circumvent the defences of bacteria (known as the 'cell envelope') in order to kill them. As our model, we use the non-pathogenic organism E. coli K-12. We have recently made two major advances in understanding how colicins translocate into E. coli (translocation is the process by which they move from the outside of the cell to the inside). First, we have isolated complexes of colicins bound to their cell surface targets, which primes them for entry into the cell, and have obtained preliminary data to suggest that we will be able to determine three dimensional structures for such translocation-competent states. Second, we have devised new fluorescence-based microscopy tools for visualizing the entry of these molecules into bacteria in real time, allowing us to address questions that have until now been impossible to investigate.The proposal has three specific objectives:1. To use crystallographic methods to determine the three dimensional structures of colicins bound to proteins that constitute the translocation machinery in E. coli;2. To use colicins impregnated with photoreactive cross-linking groups (which can be engineered genetically) as a means of trapping colicins as they pass through the cell envelope when cells are illuminated with UV light. This approach will allow us to follow the path taken by these molecules through the cell envelope and identify proteins they come into contact with;3. To exploit the microscopy tools we have developed to track colicins as they pass through the cell envelope and dissect their mechanism of entry using protein engineering.