Biofilm-protected microbial infections in skin are a serious health risk that remains to be adequately addressed. The lack of progress in developing effective treatment strategies is largely due to the transport barriers posed by the stratum corneum of the skin and the biofilm. In this work, we report on the use of Ionic Liquids (ILs) for biofilm disruption and enhanced antibiotic delivery across skin layers. We outline the syntheses of ILs, analysis of relevant physicochemical properties, and subsequent neutralization effects on two biofilm-forming pathogens: Pseudomonas aeruginosa and Salmonella enterica. Further, the ILs were also examined for cytotoxicity, skin irritation, delivery of antibiotics through the skin, and treatment of biofilms in a wound model. Of the materials examined, choline-geranate emerged as a multipurpose IL with excellent antimicrobial activity, minimal toxicity to epithelial cells as well as skin, and effective permeation enhancement for drug delivery. Specifically, choline-geranate was comparable with, or more effective than, bleach treatment against established biofilms of S. enterica and P. aeruginosa, respectively. In addition, cholinegeranate increased delivery of cefadroxil, an antibiotic, by >16-fold into the deep tissue layers of the skin without inducing skin irritation. The in vivo efficacy of choline-geranate was validated using a biofilm-infected wound model (>95% bacterial death after 2-h treatment). This work establishes the use of ILs for simultaneous enhancement of topical drug delivery and antibiotic activity.antibacterial | antimicrobial agents | antibiotic resistance | ion-pairing | formulation

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.

The siderophores of Bacillus anthracis are critical for the pathogen's proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of 1H and 13C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.

A contrathermodynamic sequence selectivity (5′-deoxyadenosine > 5′-deoxyguanosine) for UVirradiation-induced strand damage in duplex DNA containing 5-bromo-2′-deoxyuridine was reported several years ago (Saito, I.; Sugiyama, H. J. Am. Chem. Soc. 1990, 112, 6720.). In contrast, much smaller sequence selectivity was observed for similar duplexes containing 5-iodo-2′-deoxyuridine. We investigated the mechanism of UV-irradiation-induced cleavage of duplex DNA containing 5-bromo-2′-deoxyuridine (1, BrdU) and 5-iodo-2′-deoxyuridine (2, IdU) under anaerobic conditions using a variety of structural probes. The preference for UV-induced cleavage in 5′-dABrdU sequences is a confluence of at least three factors, photoinduced forward electron transfer, charge recombination, and electron migration within the DNA duplex. Our results also indicate that UV-irradiation of duplexes (32 nucleotides long) containing 5-iodo-2′-deoxyuridine results in strand scission involving initial photoinduced single electron transfer. The selectivity for 5′-dAIdU sequences is smaller than that in the analogous 5-bromo-2′-deoxyuridine duplexes and may be the result of faster dehalogenation of the initially formed 5-halopyrimidine radical anion and/or competitive direct carbon-iodine bond homolysis.5-Bromo-2′-deoxyuridine (1, BrdU) and 5-iodo-2′-deoxyuridine (2, IdU) exhibit a number of interesting and potentially useful chemical properties. 1 For instance, incorporation of these molecules in nucleic acids sensitizes the biopolymers to γ-radiolysis. 2 In addition, the 5-halopyrimidine nucleosides' sensitivity to UV-irradiation has been exploited in the application of these molecules as structural probes of protein-nucleic acid interactions and nucleic acid structure. 3,4 The enhancement of DNA damage caused by UV-irradiation of duplexes containing 1 and 2 has received considerable attention. 5-8 Approximately 10 years ago Saito and Sugiyama reported that UV-induced strand damage in duplex DNA containing 1 was highly dependent on the identity of the nucleotide bonded to the 5′-phosphate of 5-bromo-2′-deoxyuridine. 5 In contrast to previous studies, it was observed that UV-irradiated duplexes containing the sequence 5′-dABrdU exhibited significantly greater amounts of alkali-labile lesion formation than analogous molecules containing either a 2′-deoxyguanosine (dG) or 2′-deoxycytidine (dC) in place of the adjacent 2′-deoxyadenosine (dA). 9 These researchers postulated a novel mechanism involving initial photoinduced single electron transfer (PSET) from a 2′-deoxyadenosine bonded to the 5′-phosphate of 1 (Scheme 1). Preferential damage observed in 5′-dABrdU (3) sequences via PSET compared to those containing a 2′-deoxyguanosine nucleotide bonded to the 5′-phosphate of the halopyrimidine was surprising, given that electron transfer from deoxyguanosine is more favorable thermodynamically. 10 We now wish to report on studies that support this proposal, and demonstrate that the origin of this unexpected sequence selectivity for DNA damage in biopolymers ...

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