The AAHKS Best Podium Presentation Research Award: Comparing the Efficacy of Irrigation Solutions on Staphylococcal Biofilm Formed on Arthroplasty Surfaces.

BACKGROUND: A diverse array of antibacterial solutions is utilized by orthopedic surgeons in an attempt to disperse bacterial biofilm. Few studies compare these agents against biofilm grown on clinically relevant orthopedic biomaterials, such as plastic, acrylic cement, and porous titanium. METHODS: MSSA biofilm was grown on plastic 48-well plates, polymethylmethacrylate cement beads and porous Ti-6Al-4V acetabular screw caps. Antibacterial solutions were tested according to manufacturer guidance and included: isotonic saline, vancomycin (1 mg/mL), polymyxin-bacitracin (500,000 U/L-50,000 U/L), povidone-iodine 0.3%, povidone-iodine 10%, a 1:1 combination of povidone-iodine 10% & 4% hydrogen peroxide, polyhexamethylene biguanide (PHMB) and betaine 0.04%, a commercial solution containing chlorhexidine gluconate (CHG) 0.05%, and a commercial solution containing benzalkonium chloride and ethanol. Twenty four and 72-hour biofilms were exposed to solutions for 3 minutes to reproduce intraoperative conditions. Solution efficacy was measured through sonication of treated surfaces followed by counting colony forming units and validated with a resazurin assay to assess cell viability. Experiments were performed in triplicate and repeated at least once. A three-fold log reduction in CFU counts versus controls was considered as a measure of solution efficacy. RESULTS: Saline, vancomycin and polymyxin-bacitracin were ineffective compared to other solutions against planktonic MSSA. Povidone-iodine 10% and a 1:1 solution of povidone-iodine 10% and 4% hydrogen peroxide were the only effective solutions against biofilm across all three surfaces and time points. CONCLUSION: Commercial antibacterial solutions vary significantly in their efficacy against MSSA biofilm. Efficacy globally decreased as biofilm maturity increased. Increased solution cost did not confer increased efficacy.

Accessibility of substituted cysteines in TM2 and TM10 transmembrane segments in the Plasmodium falciparum equilibrative nucleoside transporter PfENT1.

Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophic. They import purines via an equilibrative nucleoside transporter (ENT). In P. falciparum, the most virulent species, the equilibrative nucleoside transporter 1 (PfENT1) represents the primary purine uptake pathway. This transporter is a potential target for the development of antimalarial drugs. In the absence of a high-resolution structure for either PfENT1 or a homologous ENT, we used the substituted cysteine accessibility method (SCAM) to investigate the membrane-spanning domain structure of PfENT1 to identify potential inhibitor-binding sites. We previously used SCAM to identify water-accessible residues that line the permeation pathway in transmembrane segment 11 (TM11). TM2 and TM10 lie adjacent to TM11 in an ab initio model of a homologous Leishmania donovani nucleoside transporter. To identify TM2 and TM10 residues in PfENT1 that are at least transiently on the water-accessible transporter surface, we assayed the reactivity of single cysteine-substitution mutants with three methanethiosulfonate (MTS) derivatives. Cysteines substituted for 12 of 14 TM2 segment residues reacted with MTS-ethyl-ammonium-biotin (MTSEA-biotin). At eight positions, MTSEA-biotin inhibited transport, and at four positions substrate transport was potentiated. On an α helical wheel projection of TM2, the four positions where potentiation occurred were located in a cluster on one side of the helix. In contrast, although MTSEA-biotin inhibited 9 of 10 TM10 cysteine-substituted mutants, the reactive residues did not form a pattern consistent with either an α helix or ß sheet. These results may help identify the binding site(s) of PfENT1 inhibitors.

Substrate and Inhibitor Specificity of the Plasmodium berghei Equilibrative Nucleoside Transporter Type 1.

Malaria is a critical public health issue in the tropical world, causing extensive morbidity and mortality. Infection by unicellular, obligate intracellular Plasmodium parasites causes malaria. The emergence of resistance to current antimalarial drugs necessitates the development of novel therapeutics. A potential novel drug target is the purine import transporter. Because Plasmodium parasites are purine auxotrophic, they must import purines from their host to fulfill metabolic requirements. They import purines via equilibrative nucleoside transporter 1 (ENT1) homologs. Recently, we used a yeast-based high-throughput screen to identify inhibitors of the P. falciparum ENT1 (PfENT1) that kill P. falciparum parasites in culture. P. berghei infection of mice is an animal model for human malaria. Because P. berghei ENT1 (PbENT1) shares only 60% amino acid sequence identity with PfENT1, we sought to characterize PbENT1 and its sensitivity to our PfENT1 inhibitors. We expressed PbENT1 in purine auxotrophic yeast and used radiolabeled substrate uptake to characterize its function. We showed that PbENT1 transports both purines and pyrimidines. It preferred nucleosides compared with nucleobases. Inosine (IC50 = 3.7 µM) and guanosine (IC50 = 21.3 µM) had the highest affinities. Our recently discovered PfENT1 inhibitors were equally effective against both PbENT1- and PfENT1-mediated purine uptake. The PfENT1 inhibitors are at least 10-fold more potent against PfENT1 than human hENT1. They kill P. berghei parasites in 24-hour ex vivo culture. Thus, the P. berghei murine malaria model may be useful to evaluate the efficacy of PfENT1 inhibitors in vivo and their therapeutic potential for treatment of malaria.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5-HT3A ) that the interaction with RIC-3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC-3 attenuated serotonin-induced currents in 5-HT3A wild-type channels, but not in functional 5-HT3A glvM3M4 channels that have the 115-amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand-gated ion channel (GLIC) to contain the 5-HT3A -ICD confers sensitivity to RIC-3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC-3 protein expression in X. laevis oocytes. We purified RIC-3 to homogeneity after expression in Echericia coli. By using heterologously over-expressed and purified RIC-3 and the chimera consisting of the 5-HT3A -ICD and the extracellular and transmembrane domains of GLIC in pull-down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5-HT3 A R that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3 A R-ICD and RIC-3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC-5-HT3A -ICD chimera interacts with RIC-3. We hypothesized that pentameric ligand-gated ion channels (pLGICs) associate directly with the chaperone protein RIC-3 (resistance to inhibitors of cholinesterase type 3), and that the interaction does not require other protein factors. We found that the two proteins indeed interact directly, that the pLGIC intracellular domain is required for the effect, and that pLGICs in their pentameric form associate with RIC-3. These results provide the basis for future studies aimed at investigating which motifs provide the interaction surfaces, and at delineating the mechanism(s) of RIC-3 modulation of functional pLGIC surface expression.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.