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1.
J Imaging ; 9(8)2023 Aug 15.
Article in English | MEDLINE | ID: mdl-37623692

ABSTRACT

SIGNIFICANCE: The development of an imaging technique to accurately identify biofilm regions on tissues and in wounds is crucial for the implementation of precise surface-based treatments, leading to better patient outcomes and reduced chances of infection. AIM: The goal of this study was to develop an imaging technique that relies on selective trypan blue (TB) staining of dead cells, necrotic tissues, and bacterial biofilms, to identify biofilm regions on tissues and wounds. APPROACH: The study explored combinations of ambient multi-colored LED lights to obtain maximum differentiation between stained biofilm regions and the underlying chicken tissue or glass substrate during image acquisition. The TB imaging results were then visually and statistically compared to fluorescence images using a shape similarity measure. RESULTS: The comparisons between the proposed TB staining method and the fluorescence standard used to detect biofilms on tissues and glass substrates showed up to 97 percent similarity, suggesting that the TB staining method is a promising technique for identifying biofilm regions. CONCLUSIONS: The TB staining method demonstrates significant potential as an effective imaging technique for the identification of fluorescing and non-fluorescing biofilms on tissues and in wounds. This approach could lead to improved precision in surface-based treatments and better patient outcomes.

2.
J Inorg Biochem ; 246: 112294, 2023 09.
Article in English | MEDLINE | ID: mdl-37356379

ABSTRACT

Redox-active azothioformamides (ATFs) contain an NNCS 1,3-heterodiene motif typically found in other molecular subclasses that exhibit a wide range of cytotoxic and anti-neoplastic effects, either alone or as chelation complexes with various metals. For this study, a small library of ATF compounds was synthesized and tested across a range of microbes, fungi, and cancer cell lines for biological activity, both alone and as metal chelates of copper(I) and silver(I) salts. Alone, the ATF compounds exhibited little antimicrobial activity, but all inhibited the cell growth of A549 lung carcinoma cells (IC50 values of 1-6 µM). As copper(I) and silver(I) coordination complexes, several of the ATFs showed antimicrobial activity against gram positive Staphylococcus aureus and Bacillus subtilis cells (IC50 âˆ¼ 5-20 µM) and the fungi Candida albicans (IC50 âˆ¼ 8-12 µM); as well as cytotoxicity against both lung carcinoma A549 cells and lymphoblastic leukemia K562 cells.


Subject(s)
Anti-Infective Agents , Antineoplastic Agents , Carcinoma , Coordination Complexes , Humans , Copper/pharmacology , Anti-Infective Agents/pharmacology , Anti-Infective Agents/metabolism , Silver/pharmacology , Cell Line , Coordination Complexes/pharmacology , Chelating Agents/pharmacology , Fungi , Microbial Sensitivity Tests , Antineoplastic Agents/pharmacology , Anti-Bacterial Agents/pharmacology
3.
IEEE Trans Radiat Plasma Med Sci ; 6(5): 619-625, 2022 May.
Article in English | MEDLINE | ID: mdl-36338575

ABSTRACT

Cold atmospheric pressure plasma (CAP) treatment has been shown to kill bacteria and remove bacterial biofilms from surfaces. Here we report the etch capacity of a linear discharge CAP device on Pseudomonas fluorescens biofilms. A 21 kHz, 1.4 kV RMS AC voltage applied to the CAP electrodes generated a hydrated Ar plasma between the plates, with the gas flow directing the plasma species toward the biological sample, causing both bacterial killing and etching of the biofilm. Typical discharge currents for a 2.4 cm long, 0.6 mm wide linear discharge device were 1-4.4 mA. Hydrated Ar flow gas was critical for removal of biofilm from a stainless steel substrate, while both hydrated and dry Ar + O2, Ar + air, O2 only, and air only flow gas mixtures did not cause etching at equivalent or greater discharge current intensities. A biofilm etch rate of > 2 µm/min was achieved, provided the plasma discharge was within 1-2 mm of the substrate surface and used a hydrated Ar gas flow of at least 5 LPM.

4.
Article in English | MEDLINE | ID: mdl-34024956

ABSTRACT

Cold atmospheric pressure plasma (CAP) has been shown to kill bacteria and remove biofilms. Here we report the development of a unique CAP array device consisting of a parallel stack of eight linear-discharge plasma elements that create a ~ 5 cm2 (2.4 cm × 2 cm) treatment area. The CAP device is fabricated from Low Temperature Co-fired Ceramic (LTCC) layers to create 24 mm long linear-discharge channels (500 µm gap) with embedded opposing silver metal electrodes. A 20 kHz AC voltage (0.5-5 kV) applied to the electrodes generates an Ar/O2 plasma between the plates, with the gas flow directing the reactive species toward the biological sample (biofilms, etc.) to affect the antimicrobial treatment. External ballast resistors were used to study discharge uniformity in the stacked array elements and internal thick film ballast resistors (≈150 kΩ) were developed to create a fully integrated device. Typical element discharge currents were 1-2.5 mA with the total array current tested at 20 mA to provide optimal device uniformity. The plasma discharge was further shown to produce reactive hydrogen peroxide and exert antimicrobial effects on Pseudomonas biofilms and Salmonella contaminated eggshell samples, with >99% of the bacterial cells killed with less than 60 seconds of plasma exposure.

5.
RSC Adv ; 11(56): 35425-35435, 2021 Oct 28.
Article in English | MEDLINE | ID: mdl-35493168

ABSTRACT

Organic semiconductors, including graphitic carbon nitride (g-C3N4, CN), represent an important class of materials for the development of novel antimicrobial or biomedical technologies. Of principal interest is the ability of these materials to catalyze the reduction of elemental oxygen to generate reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Here, we describe the fabrication of photoactive van der Waals heterojunctions incorporating 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTCDA) and CN. The composite heterojunction systems were characterized by a combination of physical (TEM, SEM, pXRD), spectroscopic (FT-IR, XPS, DRUV, photoluminescence, TCSPC) and kinetic experiments. Electronic interactions between the two components of the heterojunction increase the rate of photochemical production of H2O2 from elemental oxygen by 410%, relative to samples of pure CN. Mechanistic analysis reveals that interaction of NTCDA with the surface of CN modifies the mechanism of H2O2 formation in the heterojunction photocatalysts. The photochemical production of H2O2 by irradiation of the most active heterojunction composition is sufficient to reduce the viability of E. coli O157:H7, S. aureus and Ps. aeruginosa PAO1 by 99%. Importantly, H2O2 production by the NTCDA/CN heterojunctions suppresses Ps. aeruginosa biofilm formation, even at light exposure doses that had a lesser impact on overall planktonic cell growth.

6.
Pathogens ; 9(9)2020 Aug 20.
Article in English | MEDLINE | ID: mdl-32825529

ABSTRACT

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5'-deoxy-5'-iodoadenosine (IADO) and 5'-deoxy-5'-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.

7.
Int J Latest Trends Eng Technol ; 15(3): 036-41, 2020 Jan.
Article in English | MEDLINE | ID: mdl-32219149

ABSTRACT

A cold atmospheric pressure plasma device was developed using two parallel plates of Low Temperature Co-fired Ceramic with embedded electrodes. The 2.4 cm wide by 1 mm deep plasma discharge operates at 20 kHz with a 2-5 kV AC drive signal across a 0.25 mm gap. Mixed Argon/oxygen plasmas were directed between the plates to flow toward a bacterial biofilm sample for treatment. Results showed that at 4-5 kV the plasma etched away a bacterial biofilm on glass in 10 minutes. In addition, we showed that short plasma treatments rapidly killed biofilm resident bacteria with ED90 values of <15 s.

8.
ACS Appl Bio Mater ; 3(3): 1681-1689, 2020 Mar 16.
Article in English | MEDLINE | ID: mdl-33738440

ABSTRACT

Free-standing, composite hydrogels containing the visible-light responsive metal-free semiconductor graphitic carbon nitride (g-C3N4) as an integral component have been fabricated by direct casting techniques. At 0.67% g-C3N4 loading, intermolecular interactions between the semiconductor particles and the PVA polymer chains enhance both the mechanical and photophysical properties of the resulting hydrogels. In contrast, much higher g-C3N4 loadings of 3.3 or 6.7% g-C3N4 resulted in growth of the average semiconductor particle size and reduction in interactions between the incorporated photocatalyst and the PVA chains. The increased dimensions of the g-C3N4 semiconductor particles had the effect of compromising the mechanical properties of the composite system and reducing the lifetime of photogenerated charge carriers. However, the close proximity of g-C3N4 particles that is realized at increased semiconductor loading densities improves the absorption cross section of the material, resulting in an overall improvement in the photocatalytic activity of the material. Application of visible radiation caused all of the composite hydrogels to generate hydrogen peroxide (H2O2) at catalytic rates of 0.9-2.5 µM/min, while H2O2 decomposition rates remained similar across the different preparations. In studies to examine antimicrobial performance, irradiation of 6.7% g-C3N4/PVA hydrogel samples with visible radiation (400 ≤ λ ≤ 800 nm) generated sufficient H2O2 to significantly reduce both the viable planktonic cell population and biofilm formation in cultures of Pseudomonas aeruginosa.

9.
Biochim Biophys Acta Gen Subj ; 1864(1): 129455, 2020 01.
Article in English | MEDLINE | ID: mdl-31669585

ABSTRACT

BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4 µg/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Borrelia burgdorferi/enzymology , Lyme Disease/drug therapy , N-Glycosyl Hydrolases/genetics , Borrelia burgdorferi/drug effects , Borrelia burgdorferi/pathogenicity , Deoxyadenosines/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic/drug effects , Humans , Lyme Disease/enzymology , Lyme Disease/microbiology , N-Glycosyl Hydrolases/antagonists & inhibitors , S-Adenosylhomocysteine/metabolism , Thionucleosides/metabolism
10.
J Colloid Interface Sci ; 505: 910-918, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-28675870

ABSTRACT

In this manuscript, we describe the fabrication of photoactive biocidal or sporicidal films from urea-derived graphitic carbon nitride (u-g-C3N4). Co-deposited films of u-g-C3N4 and Escherichia coli O157:H7 (IC50=14.1±0.2mJ) or Staphylococcus aureus (methicillin resistant IC50=33.5±0.2mJ, methicillin sensitive IC50=42.7±0.5mJ) demonstrated significantly enhanced bactericidal behavior upon administration of visible radiation (400nm≤λ≤426nm). In all cases, complete eradication of the microbial sample was realized upon administration of 100mJ of visible radiation, while no antimicrobial activity was observed for non-irradiated samples. In contrast, Bacillus anthracis endospores were more resistant to u-g-C3N4 mediated killing with only a ca. 25% reduction in spore viability when treated with a 200mJ dose of visible radiation. Characterization of u-g-C3N4 reveals that the improved activity results from enhancements of both the surface area and reduction potential of the material's conduction band edge, coupled with fast injection of charge carriers into localized states and a decline in radiative recombination events. The results of this study demonstrate that g-C3N4-based materials offer a viable scaffold for the development of new, visible light driven technologies for controlling potentially pathogenic microorganisms.


Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/growth & development , Graphite/chemistry , Nitriles/chemistry , Spores, Bacterial/growth & development , Staphylococcus aureus/growth & development , Urea/chemistry , Anti-Bacterial Agents/chemistry , Catalysis , Escherichia coli/drug effects , Light , Photochemical Processes , Spores, Bacterial/drug effects , Staphylococcus aureus/drug effects
11.
Toxicol Appl Pharmacol ; 311: 42-51, 2016 Nov 15.
Article in English | MEDLINE | ID: mdl-27693115

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8weeks with 0.5ml/kg carbon tetrachloride (CCl4). TCDD (20µg/kg) or peanut oil (vehicle) was administered once a week during the last 2weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-ß1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.


Subject(s)
Hepatic Stellate Cells/drug effects , Inflammation/chemically induced , Liver Cirrhosis/chemically induced , Polychlorinated Dibenzodioxins/toxicity , Animals , Carbon Tetrachloride/toxicity , Collagen Type I/metabolism , Collagenases/metabolism , Hepatic Stellate Cells/metabolism , Liver/drug effects , Liver/enzymology , Liver/metabolism , Liver Cirrhosis/metabolism , Male , Matrix Metalloproteinase 13/metabolism , Mice , Mice, Inbred C57BL , Severity of Illness Index
12.
RSC Adv ; 6(48): 42240-42248, 2016.
Article in English | MEDLINE | ID: mdl-27672437

ABSTRACT

Photoactive films derived from nanostructured samples of metal-free, intermediate band gap semiconductor graphitic carbon nitride (ns-g-C3N4) have been synthesized and characterized for their particle properties and antimicrobial activity. Physical characterization reveals that these materials are composed of discrete nanoparticles whose dimensions range from 200 nm to 700 nm. Investigation of the photochemical reactivity of ns-g-C3N4 using coumarin-3-carboxylic acid (3-CCA) indicates that this material produces reactive oxygen species (ROS) under visible radiation. When irradiated with 0.31J visible light, ns-g-C3N4-based materials reduced the viability of both gram-negative Escherichia coli O157:H7 and gram-positive Staphylococcus aureus by approximately 50%. Nearly complete inactivation of both strains of microorganisms was achieved upon administration of a 0.62J dose of visible radiation. Importantly, no biocidal activity was observed for non-irradiated samples, indicating that the g-C3N4-derived films are not inherently toxic in the absence of visible light. The results of this study suggest that materials and, by extention, films and coatings derived from g-C3N4 may present a novel route for controlling pathogenic microorganisms on surfaces in the environment, and could be useful in reducing incidents of hospital-acquired infections.

13.
Toxicology ; 344-346: 26-33, 2016 Feb 17.
Article in English | MEDLINE | ID: mdl-26860701

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-ß. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.


Subject(s)
Cell Proliferation/drug effects , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Polychlorinated Dibenzodioxins/toxicity , Cell Line , Cell Proliferation/physiology , Humans , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/metabolism
14.
Bioorg Med Chem ; 23(23): 7378-85, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26541587

ABSTRACT

Two synthetic aziridinomitosenes (AZMs), Me-AZM and H-AZM, structurally related to mitomycin C (MC) were evaluated for their anticancer activity against six cancer cell lines (HeLa, Jurkat, T47D, HepG2, HL-60, and HuT-78) and tested for their DNA-modifying abilities in Jurkat cells. Cytotoxicity assays showed that Me-AZM is up to 72-fold and 520-fold more potent than MC and H-AZM, respectively. Me-AZM also demonstrated increased DNA modification over MC and H-AZM in alkaline COMET and Hoechst fluorescence assays that measured crosslinks in cellular DNA. Me-AZM and H-AZM treatment of Jurkat cells was found to sponsor significant DNA-protein crosslinks using a K-SDS assay. The results clearly indicate that the AZM C6/C7 substitution pattern plays an important role in drug activity and supports both DNA-DNA and DNA-protein adduct formation as mechanisms for inducing cytotoxic effects.


Subject(s)
Antineoplastic Agents/pharmacology , Cross-Linking Reagents/pharmacology , DNA/metabolism , Mitomycins/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Comet Assay , Cross-Linking Reagents/chemistry , DNA Adducts/metabolism , Humans , Mitomycins/chemistry , Structure-Activity Relationship
15.
Toxins (Basel) ; 6(4): 1397-418, 2014 Apr 22.
Article in English | MEDLINE | ID: mdl-24759174

ABSTRACT

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.


Subject(s)
Antibodies, Viral/blood , Cholera Toxin/immunology , Immunoglobulin G/blood , Viral Envelope Proteins/immunology , West Nile Virus Vaccines/immunology , West Nile virus/immunology , Adjuvants, Immunologic/administration & dosage , Administration, Intranasal , Animals , Antibody-Dependent Cell Cytotoxicity , Chlorocebus aethiops , Cholera Toxin/administration & dosage , Cholera Toxin/genetics , Complement Activation , Female , Immunization Schedule , Immunization, Secondary , Mice, Inbred BALB C , Recombinant Fusion Proteins/immunology , Time Factors , Vaccines, Synthetic/immunology , Vero Cells , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/genetics , West Nile Virus Vaccines/administration & dosage , West Nile Virus Vaccines/genetics , West Nile virus/genetics
16.
Mol Microbiol ; 79(1): 7-20, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21166890

ABSTRACT

The importance of methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S-adenosylmethionine (SAM)-mediated methylation reactions, and also produces the universal quorum-sensing signal, autoinducer-2 (AI-2). SAM is also essential for synthesis of polyamines, N-acylhomoserine lactone (autoinducer-1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5'-deoxyadenosine (5'dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad-spectrum antimicrobials.


Subject(s)
Bacteria/enzymology , Bacteria/metabolism , N-Glycosyl Hydrolases/metabolism , Purine-Nucleoside Phosphorylase/metabolism , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/metabolism , Adenine/metabolism , Bacteria/growth & development , Biofilms/growth & development , Homoserine/analogs & derivatives , Homoserine/metabolism , Lactones/metabolism , Metabolic Networks and Pathways , Methionine/metabolism , Models, Biological , Polyamines/metabolism , S-Adenosylmethionine/metabolism , Vitamins/metabolism
17.
Bioorg Med Chem ; 17(16): 5894-9, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19628399

ABSTRACT

Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.


Subject(s)
Conotoxins/chemistry , Nicotinic Antagonists/chemistry , Receptors, Nicotinic/chemistry , Amino Acid Sequence , Conotoxins/pharmacology , Humans , Molecular Conformation , Molecular Sequence Data , Nicotinic Antagonists/pharmacology , Parkinson Disease/physiopathology , Protein Binding , Protein Isoforms , Receptors, Nicotinic/metabolism
18.
J Antimicrob Chemother ; 63(6): 1163-72, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19376840

ABSTRACT

BACKGROUND: Lyme disease is the most prevalent tick-borne disease in the USA with the highest number of cases (27 444 patients) reported by CDC in the year 2007, representing an unprecedented 37% increase from the previous year. The haematogenous spread of Borrelia burgdorferi to various tissues results in multisystemic disease affecting the heart, joints, skin, musculoskeletal and nervous system of the patients. OBJECTIVES: Although Lyme disease can be effectively treated with doxycycline, amoxicillin and cefuroxime axetil, discovery of novel drugs will benefit the patients intolerant to these drugs and potentially those suffering from chronic Lyme disease that is refractory to these agents and to macrolides. In this study, we have explored 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase as a drug target for B. burgdorferi, which uniquely possesses three genes expressing homologous enzymes with two of these proteins apparently exported. METHODS: The recombinant B. burgdorferi Bgp and Pfs proteins were first used for the kinetic analysis of enzymatic activity with both substrates and with four inhibitors. We then determined the antispirochaetal activity of these compounds using a novel technique. The method involved detection of the live-dead B. burgdorferi by fluorometric analysis after staining with a fluorescent nucleic acids stain mixture containing Hoechst 33342 and Sytox Green. RESULTS: Our results indicate that this method can be used for high-throughput screening of novel antimicrobials against bacteria. The inhibitors formycin A and 5'-p-nitrophenythioadenosine particularly affected B. burgdorferi adversely on prolonged treatment. CONCLUSIONS: On the basis of our analysis, we expect that structure-based modification of the inhibitors can be employed to develop highly effective novel antibiotics against Lyme spirochaetes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Borrelia burgdorferi/drug effects , Borrelia burgdorferi/enzymology , N-Glycosyl Hydrolases/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Drug Evaluation, Preclinical , Formycins/pharmacology , Humans , Microbial Viability
19.
J Mol Biol ; 378(1): 112-28, 2008 Apr 18.
Article in English | MEDLINE | ID: mdl-18342331

ABSTRACT

5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.


Subject(s)
Arabidopsis Proteins/chemistry , Deoxyadenosines/chemistry , Purine-Nucleoside Phosphorylase/chemistry , Thionucleosides/chemistry , Amino Acid Sequence , Arabidopsis Proteins/genetics , Binding Sites , Catalysis , Crystallography, X-Ray , Hydrolysis , Kinetics , Molecular Sequence Data , Protein Conformation , Purine-Nucleoside Phosphorylase/genetics , Substrate Specificity
20.
BMC Struct Biol ; 7: 70, 2007 Oct 25.
Article in English | MEDLINE | ID: mdl-17961230

ABSTRACT

BACKGROUND: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. RESULTS: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPgammaS and MTR has been determined at 1.9 A resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. CONCLUSION: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.


Subject(s)
Arabidopsis/enzymology , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Bacillus subtilis/enzymology , Binding Sites , Conserved Sequence , Magnesium/chemistry , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Nucleotides/chemistry , Nucleotides/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Solvents , Structural Homology, Protein
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