Ways to ESKAPE: Identification of potential targets of the cell envelope of Pseudomonas aeruginosa for anti-virulence drug development

Abstract

Pseudomonas aeruginosa (Pa) is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant Pa strains is rising. Therefore, new strategies for therapy are urgently needed. Hence, in this study two different strategies were used to identify possible targets for the development of anti-infective drugs. One strategy was to investigate proteins involved in the outer membrane assembly in order to disturb the outer membrane. The assembly of outer membrane proteins is managed by the BAM complex and periplasmic chaperones like SurA. Hence, in the first part of this study, deletion mutants of the BAM complex components BamB and BamC and of chaperones HlpA and SurA were generated and investigated. The constructed deletion mutants were analysed regarding their outer membrane permeability, outer membrane composition, morphology and their virulence in the Galleria mellonella infection model. The most profound effects were found upon depletion of SurA, including increased membrane permeability, enhanced sensitivity to antibiotics and reduced virulence in the Galleria mellonella infection model. More importantly, the SurA depletion in a multi-drug resistant Pa strain resensitized the strain to treatment with clinically relevant antibiotics. Depletion of BamB showed less changes in the outer membrane composition and hence less sensitivity against antibiotics and no relevant phenotypical effects. In addition, deletion of bamC or hlpA did not present any notable effects and none of these deletion mutants could prolong the survival of the Galleria mellonella larvae compared to infection with the wildtype strain. Thus, the data underline the importance of SurA in outer membrane biogenesis in Pa. The other strategy was to target non-essential genes that are not directly responsible for resistance mechanisms but essential under exposure to therapeutic antibiotic concentrations. Therefore, a high- density transposon (Tn) library in a multidrug-resistant Pa isolate was constructed and exposed to cefepime and meropenem. The depletion of Tn insertions was measured and revealed several interesting genes involved in peptidoglycan metabolism. Validation of in frame deletion mutants by measuring ampC expression, AmpC activity and antibiotic susceptibility confirmed their relevance regarding ß-lactam reistance and their role as promising targets for anti-infective drugs. To sum up, by using two different strategies, proteins of the cell envelope or proteins involved in the cell envelope assembly of Pa were identified, which may serve as potential targets to develop anti-infective drugs in order to combat the rise of multi-drug resistant pathogens.