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Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis
Title
Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis.
Creator
Rodrigues Felix, Carolina, Rohde, Kyle, Jewett, Mollie, Self, William, Phanstiel, Otto, University of Central Florida
Abstract / Description
Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M....
Show moreTuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.
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Date Issued
2017
Identifier
CFE0007294, ucf:52172
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007294
Synthesis and Characterization of Antimicrobial Non-Color Forming Silica-Silver Nanocomposite
Title
Synthesis and Characterization of Antimicrobial Non-Color Forming Silica-Silver Nanocomposite.
Creator
Bazata, Joshua, Santra, Swadeshmukul, Moore, Sean, Jewett, Travis, University of Central Florida
Abstract / Description
Silver has been utilized for its antimicrobial properties for thousands of years in a variety of fields, extending the shelf life of food and water, rendering eating utensils sanitary, and more recently in biomedical applications such as silver based antiseptic creams. While effective as an antimicrobial agent at very low concentrations ((&)#181;g/mL), silver imparts a strong color to objects it is incorporated into, due to its high plasmonic efficiency. The goal of this study was to...
Show moreSilver has been utilized for its antimicrobial properties for thousands of years in a variety of fields, extending the shelf life of food and water, rendering eating utensils sanitary, and more recently in biomedical applications such as silver based antiseptic creams. While effective as an antimicrobial agent at very low concentrations ((&)#181;g/mL), silver imparts a strong color to objects it is incorporated into, due to its high plasmonic efficiency. The goal of this study was to determine if incorporating silver nanoparticles into a silica matrix could reduce or eliminate the plasmonic signal, while retaining the antimicrobial effects of the silver nanoparticles.Citrate capped silver nanoparticles (AgNP) were synthesized using a borohydride reduction method as outlined by Zheng et. al., and incorporated into silica nanoparticles using a method adapted from Fleger et. al. To test the antimicrobial efficacy of these synthesized silica coated silver nanoparticles (SiAgNP), minimum inhibitory concentration testing at three time points, 1, 4, and 8 hours, was carried out against E. coli and S. aureus using broth microdilution and Alamar Blue as an indicator of microbial growth. Efficacy was judged against uncoated AgNP and aqueous silver nitrate (AgNO3) solutions at equivalent Ag concentrations. Silica nanoparticles (SiNP) were utilized as a negative control. Further antimicrobial characterization using a bacterial viability assay revealed a time dependent killing trend in the SiAgNP, suggesting a controlled release of Ag+ from within the silica matrix. Efficacy of the SiAgNP was determined to fall between the most effective antimicrobial form of silver tested, AgNO3, and least effective, AgNP. However, the SiAgNP material exhibited no visible plasmon peak when UV-Visible spectrophotometric readings were taken, as well as remaining colorless when coated onto a ceramic substrate. Zeta potential revealed a high degree of colloidal stability of the SiAgNP. TEM imaging studies were carried out, verifying the presence of Ag within and on the silica nanoparticles, as well as the crystalline structure of the uncoated AgNP. It was determined that coating AgNP synthesized through borohydride reduction with silica through a St(&)#246;ber synthesis mechanism yields a material with enhanced antimicrobial effects compared to AgNP, but with no detectable plasmon signal, effectively producing a non-color forming silver based antimicrobial.
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Date Issued
2015
Identifier
CFE0006208, ucf:51097
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006208
CERIUM OXIDE NANOPARTICLES FOR THE DETECTION OF ANTIMICROBIAL RESISTANCE
Title
CERIUM OXIDE NANOPARTICLES FOR THE DETECTION OF ANTIMICROBIAL RESISTANCE.
Creator
Noll, Alexander, Perez, J. Manuel, University of Central Florida
Abstract / Description
The rise of antimicrobial resistance demands the development of more rapid screening methods for the detection of antimicrobial resistance in clinical samples to both give the patient the proper treatment and expedite the treatment of patients. Cerium oxide nanoparticles may serve a useful role in diagnostics due to their ability to exist in a mixed valence state and act as either oxidizing agents or reducing agents. Considering that cerium oxide nanoparticles have been shown to shift in...
Show moreThe rise of antimicrobial resistance demands the development of more rapid screening methods for the detection of antimicrobial resistance in clinical samples to both give the patient the proper treatment and expedite the treatment of patients. Cerium oxide nanoparticles may serve a useful role in diagnostics due to their ability to exist in a mixed valence state and act as either oxidizing agents or reducing agents. Considering that cerium oxide nanoparticles have been shown to shift in absorbance upon oxidation, a useful method of antimicrobial resistance detection could be based on the oxidation of cerium oxide nanoparticles. Herein, an assay is described whereby cerium oxide nanoparticle oxidation is a function of glucose metabolism of bacterial samples in the presence of an antimicrobial agent. Cerium oxide nanoparticles were shown to have an absorbance in the range of 395nm upon oxidation by hydrogen peroxide whereas mixed valence cerium oxide nanoparticles lacked an absorbance around 395nm. In the presence the hydrogen peroxide-producing glucose oxidase and either increasing concentrations of glucose or bacterial medium supplemented with increasing concentrations of glucose, cerium oxide nanoparticles were shown to increase in absorbance at 395nm. This oxidation assay was capable of measuring differences in the absorbance of E. coli and S. aureus samples grown in the presence of inhibitory and non-inhibitory concentrations of ampicillin in as little as six hours. Therefore, this cerium oxide nanoparticle oxidation assay may be very useful for use in clinical laboratories for the detection of antimicrobial resistance due to the relatively low cost, no requirement for specialized equipment and, most importantly, the reduced incubation time of the assay to as little as six hours compared to current gold standard antimicrobial resistance detection methods that require 24 hours. This assay may thus also help partially circumvent the issue of knowledge of antimicrobial resistance in infected patients before prescribing improper regimens.
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Date Issued
2011
Identifier
CFH0003760, ucf:44780
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0003760
Mixed Valence Copper(Cu)/Silica Nanocomposite: Synthesis, Characterization and Systematic Antimicrobial Studies
Title
Mixed Valence Copper(Cu)/Silica Nanocomposite: Synthesis, Characterization and Systematic Antimicrobial Studies.
Creator
Young, Mikaeel, Santra, Swadeshmukul, Self, William, Moore, Sean, University of Central Florida
Abstract / Description
Copper (Cu) compounds are widely used as effective agricultural bactericides. Continuous use of these materials has led to Cu accumulation in soil over time. The United States Environmental Protection Agency (US EPA) is concerned about potential Cu contamination in the environment. Improving biocidal efficacy of Cu is an attractive alternative, allowing reduction of Cu amount per application. In this research, we focused on making water-soluble mixed-valence Copper/Silica composite nanogel ...
Show moreCopper (Cu) compounds are widely used as effective agricultural bactericides. Continuous use of these materials has led to Cu accumulation in soil over time. The United States Environmental Protection Agency (US EPA) is concerned about potential Cu contamination in the environment. Improving biocidal efficacy of Cu is an attractive alternative, allowing reduction of Cu amount per application. In this research, we focused on making water-soluble mixed-valence Copper/Silica composite nanogel (CuSiNG) material. The objective is to improve the efficacy of Cu by manipulating Cu valence states. It has been shown in the literature that Cu (0) and Cu (I) states are more potent that Cu (II) states in terms of their antimicrobial efficacy. It is hypothesized that mixed valence Cu will exhibit improved efficacy over Cu (II). A water-soluble mixed valence Cu/silica nanogel (MV-CuSiNG) composite has been synthesized and characterized. Structure, morphology, crystallinity and composition of the MV-CuSiNG material was characterized using High-Resolution Transmission Electron Microscopy (HRTEM), HRTEM Selected Area Electron Diffraction (SAED) and X-ray Photoelectron Spectroscopy (XPS). Amount of Cu loading in MV-CuSiNG composite material was estimated by Atomic Absorption Spectroscopy (AAS). To confirm presence of Cu (I) in the MV-CuSiNG material, Neocuproine (Nc, a Cu (I) specific chelator) assay was used. Antimicrobial efficacy of MV-CuSiNG and CuSiNG was evaluated against X.alfalfae, B.subtilis and E.coli using Kocide(&)#174; 3000 ((")Insoluble Cu (II)(") compound), Copper sulfate ((")Soluble Cu (II)(") compound) and Cuprous chloride (Copper (I) compound) as positive controls and silica (")seed(") particles (without Cu loading) as negative control. Antimicrobial studies included observing bacterial growth inhibition and determining the Minimum Inhibitory Concentration (MIC). Improved antimicrobial efficacy was observed in MV-CuSiNG when compared to CuSiNG and other controls. For the assessment of plant safety of MV-CuSiNG and CuSiNG materials, phytotoxicity studies were conducted using Vinca sp and Hamlin orange under environmental conditions. It was observed that MV-CuSiNG material was safe to plants at commercially used (standard) spray application rate.
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Date Issued
2013
Identifier
CFE0005282, ucf:50550
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005282
Development of S-nitroso-N-acetylpenicillamine (SNAP) Impregnated Medical Grade Polyvinyl Chloride for Antimicrobial Medical Device Interfaces
Title
Development of S-nitroso-N-acetylpenicillamine (SNAP) Impregnated Medical Grade Polyvinyl Chloride for Antimicrobial Medical Device Interfaces.
Creator
Feit, Corbin, Brisbois, Elizabeth, Vaidyanathan, Raj, Kuebler, Stephen, University of Central Florida
Abstract / Description
In the clinical setting, polyvinyl chloride (PVC) accounts for 25% of all polymers used in medical device applications. However, medical devices fabricated with PVC, such as endotracheal tubes, extracorporeal circuits (ECCs), or intravenous catheters suffer from thrombosis and infection. Mortality associated with hospital associated infections (HAIs) exceed 100,000 deaths each year. One method to overcome these challenges is to develop bioactive polymers with nitric oxide (NO) release. Nitric...
Show moreIn the clinical setting, polyvinyl chloride (PVC) accounts for 25% of all polymers used in medical device applications. However, medical devices fabricated with PVC, such as endotracheal tubes, extracorporeal circuits (ECCs), or intravenous catheters suffer from thrombosis and infection. Mortality associated with hospital associated infections (HAIs) exceed 100,000 deaths each year. One method to overcome these challenges is to develop bioactive polymers with nitric oxide (NO) release. Nitric oxide exhibits many physiological roles including, antibacterial, antithrombic, anti-inflammatory activity. In this study, Tygon(&)#174; PVC tubing was impregnated with a NO donor molecule, S-nitroso-N-acetylpenicillamine (SNAP), via a simple solvent-swelling-impregnation method, where polymer samples were submerged in a SNAP impregnation-solvent (methanol, acetone, plasticizer). An additional topcoat of a biocompatible CarboSil 2080A (CB) was applied to reduce SNAP leaching. The SNAP-PVC-CB were characterized for NO release using chemiluminescence, leaching with UV-Vis spectroscopy, surface characterization with scanning electron microscopy, tensile strength analysis, stability during storage and sterilization, and antimicrobial properties in vitro. The SNAP-PVC-CB exhibited NO flux of 4.29 (&)#177; 0.80 x 10-10 mol cm-2 min-1 over the initial 24 h under physiological conditions and continued to release physiological levels of NO for up 14 d (incubated in PBS at 37 (&)deg;C). The addition of CB-topcoat reduced the total SNAP leaching by 86% during incubation. Mechanical properties and surface topography remained similar to original PVC after SNAP-impregnation and application of CB-topcoat. After ethylene oxide sterilization and 1-month storage, SNAP-PVC-CB demonstrated excellent SNAP stability (ca. 90% SNAP remaining). In a 24 h antibacterial assay, SNAP-PVC reduce viable bacteria colonization (ca. 1 log reduction) of S. aureus and E. coli compared to PVC controls. This novel method for SNAP-impregnation of medical grade plasticized PVC holds great potential for improving the biocompatibility of post-fabricated PVC medical devices.
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Date Issued
2019
Identifier
CFE0007887, ucf:52782
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007887
THE ANTI-HIV-1 ACTIVITY OF HUMAN SEMINAL PLASMA
Title
THE ANTI-HIV-1 ACTIVITY OF HUMAN SEMINAL PLASMA.
Creator
Martellini, Julie, Cole, Alexander, University of Central Florida
Abstract / Description
Human immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are...
Show moreHuman immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are actively being explored. Recent trials of topical vaginal microbicides have shown that their interaction with the mucosal surfaces of the female reproductive tract as well as semen can hinder microbicide effectiveness against HIV-1 infection. Therefore, understanding the role these fluids play in HIV transmission would be critical towards developing effective antiviral prophylaxes. A recent study from our group demonstrated that human cervicovaginal secretions contained numerous cationic antimicrobial peptides and proteins, which collectively inhibited HIV-1 infection of target cells and tissues. To ascertain if human seminal plasma (SP), the main vector responsible for transmitting HIV-1, exhibited antiviral activity we utilized several anti-HIV assays in the presence or absence of minimally manipulated SP. The majority of the intrinsic anti-HIV-1 activity of SP resided in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multi-step chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP. Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" capable of enhancing HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity. Our studies demonstrate the overall antiviral activity of human SP, but there is still a critical need for effective topical vaginal microbicides that can prevent HIV-1 transmission. The synthetic human retrocyclins are cyclic antimicrobial peptides that are remarkably active against HIV-1, and are being developed as topical vaginal microbicides. Herein, we assessed whether the putative proviral SEVI was able to adversely affect the anti-HIV-1 activity of the retrocyclin analog RC-101. While SEVI alone enhanced viral infection, this effect was completely negated in the presence of RC-101. Retrocyclins such as RC-101 are inhibitors of HIV-1 entry, by preventing gp41-mediated viral fusion. Interestingly, using an HIV-1 reverse transcriptase (RT) specific assay, we also determined that RC-101 directly inhibited the activity of RT in a dose dependent manner, suggesting a secondary mechanism of viral inhibition. Our group has determined that RC-101 induces only a modest level of resistance in HIV, which may be due in part to RC-101's dual mechanisms of viral inhibition.
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Date Issued
2011
Identifier
CFE0003583, ucf:48916
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003583
RETROCYCLIN, A POTENT HIV-1 ENTRY INHIBITOR.
Title
RETROCYCLIN, A POTENT HIV-1 ENTRY INHIBITOR.
Creator
Venkataraman, Nitya, Cole, Alexander, University of Central Florida
Abstract / Description
Human immununodeficiency virus (HIV) infection is the leading cause of death due to viral infections worldwide. In the absence of an effective vaccine or consistent male condom use, there is a clear need for female-controlled preventatives such as topical vaginal microbicides. Recent attention has been focused on developing natural antimicrobial peptides, as anti-retroviral microbicides. Increasing evidence suggests that cationic antimicrobial peptides such as defensins are effective HIV-1...
Show moreHuman immununodeficiency virus (HIV) infection is the leading cause of death due to viral infections worldwide. In the absence of an effective vaccine or consistent male condom use, there is a clear need for female-controlled preventatives such as topical vaginal microbicides. Recent attention has been focused on developing natural antimicrobial peptides, as anti-retroviral microbicides. Increasing evidence suggests that cationic antimicrobial peptides such as defensins are effective HIV-1 inhibitors. Human alpha- and beta-defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins – 18 residue cyclic peptides that are potent HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclins genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection. Our studies on retrocyclin reveal that they are potential candidates to develop as topical vaginal microbicides to prevent sexual transmission of HIV-1. Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In a search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In our studies, we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1, and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation-exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were alone not active against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex and full anti-HIV-1 activity likely involves the aggregate of the cationic peptides and proteins in the acidic human vaginal fluid. Interestingly, retrocyclins retained complete anti-HIV-1 activity in the presence of human vaginal fluid. Therefore expression of retrocyclin peptides can help activate the natural defense mechanism against HIV-1. We next investigated the regulation of expression of retrocyclin (pseudo)gene. We identified a putative interferon response cluster upstream of the retrocyclin gene. The activity of this cluster was upregulated when treated with IFN-β although to a modest extent. Interestingly, the cluster also contained the binding site for an Interferon Consensus Sequence Binding Protein (ICSBP), a known repressor of the IFN inducible genes. Deletion of the ICSBP site or addition of a known inhibitor of ICSBP resulted in the increase in the activity of the cluster, indicating a role for ICSBP in the negative regulation of expression of retrocyclins. Collectively our data suggest that the expression of this ancestral gene is tightly regulated in both a positive and negative manner via the IFN response pathway.
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Date Issued
2009
Identifier
CFE0002777, ucf:48104
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002777
EXPRESSION AND CHARACTERIZATION OF ANTIMICROBIAL PEPTIDES RETROCYCLIN-101 AND PROTEGRIN-1 IN CHLOROPLASTS TO CONTROL VIRAL AND BACTERIAL INFECTIONS
Title
EXPRESSION AND CHARACTERIZATION OF ANTIMICROBIAL PEPTIDES RETROCYCLIN-101 AND PROTEGRIN-1 IN CHLOROPLASTS TO CONTROL VIRAL AND BACTERIAL INFECTIONS.
Creator
Li, Baichuan, Daniell, Henry, University of Central Florida
Abstract / Description
Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101...
Show moreRetrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC-101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC-101 and PG1 accumulated up to 32-38% and 17~26% of the total soluble protein. Both RC-101 and PG1 were cleaved from GFP by corresponding proteases in vitro and Factor Xa like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6 fold higher yield of RC 101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to TMV infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further pre-clinical studies.
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Date Issued
2010
Identifier
CFE0003199, ucf:48571
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003199
Membrane topology of a broad-spectrum resistance factor responsible for lipid modification in Enterococcus faecium.
Title
Membrane topology of a broad-spectrum resistance factor responsible for lipid modification in Enterococcus faecium.
Creator
Harrison, Jesse, Roy, Herve, Teter, Kenneth, Phanstiel, Otto, University of Central Florida
Abstract / Description
Aminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against...
Show moreAminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against osmotic stress and acidic conditions. aaPGS homologs are found in a variety of clinically relevant microorganisms, including Enterococcus faecium, which is increasingly found to be the etiologic agent of antibiotic-resistant nosocomial infections. Although the broad distribution of these virulence factors across bacterial species makes them attractive targets for therapeutic strategies, little is known about the structure of aaPGSs. Two aaPGS paralogs are found in E. faecium, one of which exhibits relaxed substrate specificity and is responsible for the transfer of Arg (R), Ala (A), and Lys (K) to PG (RakPGS). The catalytic site of RakPGS is located in the hydrophilic C-terminal domain, which is localized in the cytoplasm. The N-terminus contains an integral membrane domain that is thought to harbor flippase activity that translocates the neosynthesized aa-PG from the inner to the outer leaflet of the membrane. We are currently developing the substituted cysteine accessibility method (SCAM) and a dual-reporter fusion system, which exploits alkaline phosphatase (Pho) and ?-galactosidase (LacZ) activities, for investigating the membrane topology of RakPGS in E. faecium.
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Date Issued
2015
Identifier
CFE0006318, ucf:51566
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006318
Synthesis and Characterization of Core-Shell Zinc Silica Nanoparticles and Zinc Silica Nanogels for Agricultural Applications.
Title
Synthesis and Characterization of Core-Shell Zinc Silica Nanoparticles and Zinc Silica Nanogels for Agricultural Applications.
Creator
Berroth, Megan, Santra, Swadeshmukul, Moore, Sean, Jewett, Travis, University of Central Florida
Abstract / Description
Plant pathogens are a serious problem facing the agricultural industry today. Current methodologies use copper based biocides as the main form of defense. Unfortunately this can lead to damaging environmental effects and increased rates of antimicrobial resistance. In this study, antimicrobial activity of multiple alternative zinc-based nanoformulations were tested against three important plant pathogens: Xanthomonas alfalfae, Pseudomonas syringae, and Clavobacter michiganensis. Xanthomonas...
Show morePlant pathogens are a serious problem facing the agricultural industry today. Current methodologies use copper based biocides as the main form of defense. Unfortunately this can lead to damaging environmental effects and increased rates of antimicrobial resistance. In this study, antimicrobial activity of multiple alternative zinc-based nanoformulations were tested against three important plant pathogens: Xanthomonas alfalfae, Pseudomonas syringae, and Clavobacter michiganensis. Xanthomonas sub species cause Citrus canker, a devastating disease that affects millions of citrus trees worldwide while the latter two affect tomato crops. Materials synthesis was completed and the resulting nanoformulations were characterized by Atomic Absorption Spectroscopy, Scanning Electron Microscopy, High Resolution Transmission Electron Microscopy, and X-Ray Photoelectron Spectroscopy. The antimicrobial efficacy of the newly synthesized formulas and two commercially available products, Kocide 3000 (DuPont) and Nordox (Brandt), were determined by Minimum Inhibitory Concentration Assays followed by Bacterial Viability Assays. The subsequent data demonstrated a marketed difference in the way the antimicrobial agents acted upon the bacterial species. The core-shell zinc silica nanoparticles (C-SZnSiNP) proved to be ineffective, while the zinc silica nanogel (ZnSiNG) was as successful at killing the bacteria as the commercial products. This shows promise for a new alternative material with zinc at the forefront of the fight against plant pathogens.
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Date Issued
2015
Identifier
CFE0006209, ucf:51099
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006209