MRC DTP: Bacterial stress sensing and antibiotic action

Pseudomonas aeruginosa is an opportunistic pathogen that is naturally resistant to many antibiotics and can acquire mutations that increase its resistance even further. It is one of several Gram-neagtive pathogens listed by the World Health Organisation as critical threats to human health [1]. One of the mechanisms by which P. aeruginosa and other Gram-negative bacteria can avoid damage by antibiotics is by activating a cell envelope stress response that has several downstream effects. It generally inhibits cell growth and division, which decreases the activity of many antibiotic targets. It also changes expression of many cell envelope proteins, leading to upregulation of efflux pumps and downregulation of outer membrane porins. Together, these changes decrease the concentration of antibiotic within the cell. A major contributor to the cell envelope stress response is the AmgRS two-component system, which can be activated in response to antibiotic treatment [2]. This project will focus on developing a mechanistic understanding of how AmgS, a membrane-embedded protein that is the sensing component of the system, is able to detect cell envelope stress, and measuring the contribution of the response to antibiotic tolerance.

In this project, we will investigate the cell envelope stress sensing mechanisms of AmgS and its E. coli homologue, EnvZ. We will develop reporter strains for activation of the envelope stress response and determine conditions that activate it in Pseudomonas aeruginosa. We will also use genetic screens to identify additional genes contributing to the response [3]. Finally, we will conduct biomolecular simulations of EnvZ in realistic membrane mixture models and construct a structural model of AmgS. Questions of interest will include: 1) how do EnvZ and AmgS interact with the cytoplasmic membrane and other proteins required for the response; 2) how might these interactions be perturbed by conditions that lead to activation of the sensor; and 3) how do specific downstream results of activation of the pathway contribute to antibiotic tolerance? A long-term objective is to gain insight toward defeating this bacterial mechanism for increasing tolerance of antibiotics.