Dissecting Gram-negative envelope biogenesis

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The World Health Organization (WHO) identified antibiotic resistant pathogens as one of the biggest threats to global health, food security and development. Anyone can be affected, regardless of age or nationality. The growing number of pathogens that are resistant to current antibiotic treatments clearly signal a need to act against these rapidly adapting pathogens. Despite access to the most modern medicines and hospitals, non-treatable infections impact patients in several ways, ranging from longer hospital stays, to ultimately death. Thus, there is an urgent need to invest more resources in research on pathogens to be able to treat infections they cause. Therefore, the WHO issued a warning to act to prevent us from heading for a post-antibiotic area, where common infections and minor injuries would once again be deadly. The WHO have prioritised a list of the most concerning pathogens to encourage funders like the BBSRC and scientists to tackle the pathogens that are close to becoming untreatable. At the top of this list, classed as critical, are solely Gram-negative bacteria. This research proposal focusses on understanding how Gram-negative bacteria build one of their most important structures - their cell envelope. Furthering our knowledge about this process will help us to design strategies to overcome pathogen resistance to the antibiotics we use. The bacterial cell envelope is a multi-layered structure that protects the cell from its unpredictable and often hostile environment, including exposure to antibiotics. In particular, Gram-negative bacterial cell envelopes hold special interest because of the combined property of being both a structural element and a permeability barrier. The low permeability is conferred by the asymmetric lipid bilayer, referred to as the outer membrane, which prevents toxic compounds, including many antibiotics, from entering the cell. Defining which genes play a role in maintaining the structure and impermeability of the envelope is fundamental to understanding how bacteria protect themselves. It also helps us to find new ways to overcome this permeability barrier and to deliver antibiotics to treat infections. Despite the need for this kind of research, genome-wide screens to assay envelope integrity in Gram-negative bacteria are still missing. The work outlined in this proposal will fill this knowledge gap. I will develop a genome-wide, high-throughput assay to robustly quantify the underlying network of genes involved in Gram-negative envelope biosynthesis. The function of any gene can be studied by deleting it from the genome and analysing the consequences of its deletion (e.g. differences in responses to antibiotics). This can be done systematically using thousands of mutants of a pathogen, each mutant deficient in a single gene. I propose to use a collection of single deletion mutants to profile envelope biogenesis for the Gram-negative pathogens Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. This work will uncover the effect of each deleted gene on responses to many different antibiotics and environmental stresses. The resulting stress-response maps will provide knowledge about the uncharted mode of action of drugs and how those bacteria maintain their envelope integrity when challenged. By analysing these networks, I can identify genes that play fundamental roles in these processes. Once I have identified important genes or pathways, I will further investigate their cellular function. For this, I will use my expertise in molecular biology to understand if other genes are co-dependent on identified key players (genetic interactions) and if we can identify the protein machineries these proteins are part of (protein interactions). These observations will aid in the identification of potential drug targets and help to overcome the molecular barrier posed by the cell envelope, ultimately leading to better treatment of Gram-negative bacterial infections.