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

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

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate

Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics

The Bactericidal Fatty Acid Mimetic 2CCA-1 Selectively Targets Pneumococcal Extracellular Polyunsaturated Fatty Acid Metabolism

Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus. 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT. These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae

THCz : Small molecules with antimicrobial activity that block cell wall lipid intermediates

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials

Data from: Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1,000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis

Data.zip, Part 18 of 31

Data.zip, Part 18 of 3