Identification of antibiotic agents directed against gram-negative bacteria by exploiting interspecies communication

Abstract

Arising antibiotic resistances in bacteria pose a major global threat resulting in increased morbidi-ty, mortality and healthcare costs. Recently, the WHO stressed the need to develop new antibac-terial strategies and discover novel bactericidal compounds to turn the tides in the race against emerging treatment failures. Within the scope of this thesis, we contribute to that effort, focussing on the human pathogens Neisseria gonorrhoeae, causative of the sexually transmittable disease gonorrhea and Moraxella catarrhalis, a major player in pulmonary and middle ear infections. In a first approach we addressed the mechanism of gonococcal infection, and evaluated the gene transcription change of the pathogen upon interaction with its human epithelial receptors of the carcinoembryonic antigen related cell adhesion molecule (CEACAM) family. To observe in-teraction specific changes, we developed a cell free in vitro stimulation system to allow the bac-terium untainted contact with its human receptors CEACAM1 and 5. Transcriptomic analysis of stimulated samples revealed little to no changes upon receptor binding. This might indicate that the expression pattern is not influenced by the recognition of epithelial CEACAM-receptors or hint towards a non-changing transcription of gonococci when infecting their human host. Secondly, we prospected the potential of Pseudomonas aeruginosa, which is known to secure its dominance over other bacterial species through an intricate quorum sensing system. We found an inhibiting effect of Pseudomonas in direct growth competition and screened for poten-tial responsible candidates. Indeed, we identified several alkyl-quinolone-N-oxide agents, which conferred a remarkably strong and specific growth suppression on pathogenic Neisseria, while not impeding growth of commensals originating from the same genus. Out of these, nonyl-quinolone-N-oxide (NQNO) proofed to be the most efficient compound with a minimum inhibitory concentration of 5 µM (1.44 µg/mL). The compound is even able to supress proliferation in gon-ococcal strains which are resistant to multiple current treatment options, marking it as a promising candidate for the ambitious goals set by the WHO. Functionally, NQNO seems to influence bac-terial metabolism, as a reduction of ATP and an elevated production of reactive oxygen species was detected. The excellent cytotolerance displayed on cervix carcinoma cells paved the way for the medical use of NQNO. In vivo application in a humanized mouse model showed promising results, reducing the gonococcal load over a hundred fold compared to control treatment. To expand on this promising compound repertoire, we assessed the antimicrobial potential of PQS, the namesake of one of the pseudomonal quorum sensing systems. Indeed, PQS proved to strongly impair the growth of Moraxella catarrhalis. Through successive changes in the gen-eral scaffold of the agent we introduced optimizations resulting in compound 8 which harbours a nitrogen to sulphur substitution and an elongated alkyl chain. Compound 8 displayed a tenfold increased efficacy compared to PQS and was also able to inhibit growth of all tested clinical iso-lates of Moraxella catarrhalis. In this regard we detected an influence on the bacteria’s primary metabolism, reducing DNA, RNA, protein and ATP levels while leaving cellular integrity intact. Further, it inhibits growth substantially and lastingly already within 20 minutes after application starting at 0.5 µM (0.158 µg/mL). Comparable to NQNO, the compound does not negatively af-fect growth of commensal representatives of the genus and proofed to be well tolerated on hu-man pulmonary cells with a therapeutical window spanning two powers of magnitudes. Concluding, we can report, that we have identified and optimized two novel compounds derivat-ing from Pseudomonas aeruginosa that have ample potential in the fight against multiresistant gram-negative pathogenic bacteria.