ThIV-Desferrioxamine: characterization of a fluorescent bacterial probe.

Diversifying our ability to guard against emerging pathogenic threats is essential for keeping pace with global health challenges, including those presented by drug-resistant bacteria. Some modern diagnostic and therapeutic innovations to address this challenge focus on targeting methods that exploit bacterial nutrient sequestration pathways, such as the desferrioxamine (DFO) siderophore used by Staphylococcus aureus (S. aureus) to sequester FeIII. Building on recent studies that have shown DFO to be a versatile vehicle for chemical delivery, we show proof-of-principle that the FeIII sequestration pathway can be used to deliver a potential radiotherapeutic. Our approach replaces the FeIII nutrient sequestered by H4DFO+ with ThIV and made use of a common fluorophore, FITC, which we covalently bonded to DFO to provide a combinatorial probe for simultaneous chelation paired with imaging and spectroscopy, H3DFO_FITC. Combining insight provided from FITC-based imaging with characterization by NMR spectroscopy, we demonstrated that the fluorescent DFO_FITC conjugate retained the ThIV chelation properties of native H4DFO+. Fluorescence microscopy with both [Th(DFO_FITC)] and [Fe(DFO_FITC)] complexes showed similar uptake by S. aureus and increased intercellular accumulation as compared to the FITC and unchelated H3DFO_FITC controls. Collectively, these results demonstrate the potential for the newly developed H3DFO_FITC conjugate to be used as a targeting vector and bacterial imaging probe for S. aureus. The results presented within provide a framework to expand H4DFO+ and H3DFO_FITC to relevant radiotherapeutics (like 227Th).

Structure-Guided Biochemical Analysis of Quorum Signal Synthase Specificities.

Many bacteria use membrane-diffusible small molecule quorum signals to coordinate gene transcription in response to changes in cell density, known as quorum sensing (QS). Among these, acyl-homoserine lactones (AHL) are widely distributed in Proteobacteria and are involved in controlling the expression of virulence genes and biofilm formation in pathogens, such as Pseudomonas aeruginosa. AHL molecules are specifically biosynthesized by the cognate LuxI type AHL synthases using S-adenosylmethionine (SAM) and either acyl carrier protein (ACP)- or CoA-coupled fatty acids through a two-step reaction. Here, we characterize a CoA-dependent LuxI synthase from Rhodopseudomonas palustris that utilizes an aryl-CoA substrate that is environmentally derived, specifically p-coumaric acid. We leverage structures of this aryl-CoA-dependent synthase, along with our prior studies of an acyl-CoA-dependent synthase, to identify residues that confer substrate chain specificity in these enzymes. We test our predictions by carrying out biochemical, kinetic, and structural characterization of representative AHL signal synthases. Our studies provide an understanding of various AHL synthases that may be deployed in synthetic biological applications and inform on the design of specific small molecule therapeutics that can restrict virulence by targeting quorum signaling.

Insights into ß-ketoacyl-chain recognition for ß-ketoacyl-ACP utilizing AHL synthases.

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.