Determination of vulnerability of essential genes in Pseudomonas aeruginosa using CRISPR interference (CRISPRi)

• Applicant: Dr. Anne-Sophie Herbrüggen
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2017 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 395641632

Pseudomonas aeruginosa is a life-threatening nosocomial Gram-negative pathogen that was recently identified by the World Health Organization as a priority for the development of new antibiotics in order to combat its multidrug resistance. The identification of essential gene products provides one promising path to the identification of potential new targets for antibiotic discovery. However, essentiality alone is not a sufficient criterion to describe a promising drug target. Compounds inhibiting intracellular essential proteins often fail to have whole cell activity because they need to traverse both outer and inner membranes of Gram-negative cells. Active efflux by 12 or more RND efflux pumps of P. aeruginosa provide a particularly strong barrier for passage through the inner membrane and thus effectively prevent the accumulation of drugs in the bacterial cytosol. Thus, inhibitors that target exported proteins (proteins residing in the periplasm, inner- or outer membrane) might show higher efficacy against Gram-negative bacterial cells as long as they can enter cells through outer membrane proteins (OMPs) as is the case for some beta-lactam antibiotics. Other antibiotics such as colistin disrupt the integrity of outer membrane as part of their mode of action. Furthermore, the level of protein that needs to be inhibited in order to block growth of bacteria can vary among essential proteins. Hence, highly vulnerable essential targets are favorable drug targets, since lower concentrations of compound are needed to achieve growth inhibition. In this study, we propose to establish a CRISPR interference (CRISPRi) system in P. aeruginosa, a method that can be used to easily manipulate expression levels of essential genes in a high-throughput fashion. We will examine the vulnerability of gene products predicted to be located outside the cytosol of P. aeruginosa by carefully down-regulating their expression while monitoring cell viability, growth inhibition and the susceptibility to other antibiotics that are typically defeated by efflux or OM impermeability). Furthermore, we will characterize the morphological effects of the down-regulation of essential genes using live-cell imagining in a microfluidic chamber. The knowledge about highly vulnerable essential genes will help to prioritize targets for the development of novel antibiotics against Pseudomonas aeruginosa infections.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor John J. Mekalanos, Ph.D.