Targeting cell envelope synthesis of Streptococcus pneumoniae and microfluidic diagnostic tool development

Abstract

Evolving antibiotic resistance warrants the development of new therapeutic and diagnostic approaches as part of the strategies to secure future antibacterial therapies and preserve the compounds currently available. Work constituting the thesis characterized small molecules yielded from a screen for autolysis inducing compounds on Streptococcus pneumoniae. Two compound classes were characterized, and their targets identified.

The alkylated di-cyclohexyl carboxylic acid 2CCA-1 was identified as a fatty acid mimetic, that is incorporated into pneumococcal phospholipids via the polyunsaturated host fatty acid metabolism pathway. The formed 2CCA-1 containing lipids alter membrane fluidity, and treatment with 3 μM 2CCA-1 resulted in decreased pneumococcal viability and cell wall hydrolase mediated lysis. Deletion of the fatty acid binding protein FakB3 rendered pneumococci resistant to 2CCA-1, which could explain the inherent 2CCA-1 resistance of Staphylococcus aureus as FakB3 homologues are predominantly absent in bacteria of the Bacillales order. The involvement of the transcriptional repressor of the endogenous fatty acid synthesis machinery FabT in 2CCA-1 resistance, showed that FakB3 dependent host fatty acid incorporation is regulated depending on extracellular fatty acid availability.

The second compound class comprised analogs of 1-amino substituted Tetrahydrocarbazoles (THCz). THCz analogs are active in the low micromolar range against an array of gram-positive bacteria as well as mycobacteria, Neisseria gonorrhoeae and Moraxella catarrhalis. Mode of action studies identified the pyrophosphate moiety of undecaprenyl pyrophosphate as the minimal binding motif for THCz, which depended on the central diamino moiety for activity. THCz analogs consequently inhibited cell wall, teichoic acid and capsular biosynthesis. Reduction of the polysaccharide capsule increased pneumococcal tolerance to the compound, but resistant mutants could not be obtained.

Furthermore, we developed a microfluidic based sample preparation method for decomplexation of bacteria containing whole blood. First, blood cells were selectively lysed while preserving bacterial viability. For the reduction of the small, below micrometer sized debris, gradient acoustic focusing was developed, that allowed separation of bacteria from the blood lysate in a microfluidic channel. The so purified sample might facilitate further microfluidic downstream operations to accelerate antimicrobial susceptibility determination of the sepsis causing pathogen. In conclusion, the thesis work identifies the target of two new compounds with bactericidal activity and presents a microfluidic based method for sample preparation as tool in sepsis diagnosis.