Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus.

Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase-DNA complex crystal structure reveals unique protein-DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a small-angle X-ray scattering structure of a full-length, 665-residue NES-DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention.

Sensitized photodecomposition of organic bisphosphonates by singlet oxygen.

During efforts to stabilize metal oxide bound chromophores for photoelectrochemical applications, a novel photochemical reaction has been discovered. In the reaction, the bisphosphonate functional groups -C(PO(3)H(2))(2)(OH) in the metal complex [Ru(bpy)(2)(4,4'-(C(OH)(PO(3)H(2))(2)bpy)](2+) are converted into -COOH and H(3)PO(4). The reaction occurs by sensitized formation of (1)O(2) by the lowest metal-to-ligand charge transfer excited state(s) of [Ru(bpy)(2)(4,4'-(C(PO(3)H(2))(2)(OH))(2)(bpy))](2+)* followed by (1)O(2) oxidation of the bisphosphonate substituent. A related reaction occurs for the bisphosphonate-based drug, risedronic acid, in the presence of O(2), light, and a singlet oxygen sensitizer ([Ru(bpy)(3)](2+) or Rose Bengal).

Genetically engineered cancer models, but not xenografts, faithfully predict anticancer drug exposure in melanoma tumors.

BACKGROUND: Rodent studies are a vital step in the development of novel anticancer therapeutics and are used in pharmacokinetic (PK), toxicology, and efficacy studies. Traditionally, anticancer drug development has relied on xenograft implantation of human cancer cell lines in immunocompromised mice for efficacy screening of a candidate compound. The usefulness of xenograft models for efficacy testing, however, has been questioned, whereas genetically engineered mouse models (GEMMs) and orthotopic syngeneic transplants (OSTs) may offer some advantages for efficacy assessment. A critical factor influencing the predictability of rodent tumor models is drug PKs, but a comprehensive comparison of plasma and tumor PK parameters among xenograft models, OSTs, GEMMs, and human patients has not been performed. METHODS: In this work, we evaluated the plasma and tumor dispositions of an antimelanoma agent, carboplatin, in patients with cutaneous melanoma compared with four different murine melanoma models (one GEMM, one human cell line xenograft, and two OSTs). RESULTS: Using microdialysis to sample carboplatin tumor disposition, we found that OSTs and xenografts were poor predictors of drug exposure in human tumors, whereas the GEMM model exhibited PK parameters similar to those seen in human tumors. CONCLUSIONS: The tumor PKs of carboplatin in a GEMM of melanoma more closely resembles the tumor disposition in patients with melanoma than transplanted tumor models. GEMMs show promise in becoming an improved prediction model for intratumoral PKs and response in patients with solid tumors.

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1.

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 A crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 A out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.

Gluconate metabolism is required for virulence of the soft-rot pathogen Pectobacterium carotovorum.

Pectobacterium carotovorum is a ubiquitous soft rot pathogen that uses global virulence regulators to coordinate pathogenesis in response to undefined environmental conditions. We characterize an operon in P. carotovorum required for gluconate metabolism and virulence. The operon contains four genes that are highly conserved among proteobacteria (initially annotated ygbJKLM), one of which was misassigned as a type III secreted effector, (ygbK, originally known as hopAN1). A mutant with a deletion-insertion within this operon is unable to metabolize gluconate, a precursor for the pentose phosphate pathway. The mutant exhibits attenuated growth on the leaves of its host of isolation, potato, and those of Arabidopsis thaliana. Notably, the mutant hypermacerates potato tubers and is deficient in motility. Global virulence regulators that are responsive to cell wall pectin breakdown products and other undefined environmental signals, KdgR and FlhD, respectively, are misregulated in the mutant. The alteration of virulence mediated via changes in transcription of known global virulence regulators in our ygbJ-M operon mutant suggests a role for host-derived catabolic intermediates in P. carotovorum pathogenesis. Thus, we rename this operon in P. carotovorum vguABCD for virulence and gluconate metabolism.

Silylated precision particles for controlled release of proteins.

With the recent advances in the development of novel protein based therapeutics, controlled delivery of these biologics is an important area of research. Herein, we report the synthesis of microparticles from bovine serum albumin (BSA) as a model protein using Particle Replication in Non-wetting Templates (PRINT) with specific size and shape. These particles were functionalized at room temperature using multifunctional chlorosilane that cross-link the particles to render them to slowly-dissolving in aqueous media. Mass spectrometric study of the reaction products of diisopropyldichlorosilane with individual components of the particles revealed that they are capable of reacting and forming cross-links. Energy dispersive spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) were also used to confirm the functionalization of the particles. Cross sectional analysis using focused ion beam (FIB) and EDS proved that the functionalization occurs throughout the bulk of the particles and is not just limited to the surface. Circular dichroism data confirmed that the fraction of BSA molecules released from the particles retains its secondary structure thereby indicating that the system can be used for delivering protein based formulations while controlling the dissolution kinetics.