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(1 - 19 of 19)
- Title
- EXPRESSION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS 19KDA WITH POSTTRANSLATIONAL MODIFICATION.
- Creator
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Safavi-Khasraghi, Mitra, Naser, Saleh, University of Central Florida
- Abstract / Description
-
Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant...
Show moreDespite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.
Show less - Date Issued
- 2006
- Identifier
- CFE0001170, ucf:52851
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001170
- Title
- THE EFFECTS OF SITE-DIRECTED MUTAGENESIS ON HEMERYTHRIN-LIKE PROTEIN RV2633C.
- Creator
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Rosch, Kelly M, Self, William, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project...
Show moreMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project is to identify specific residues outside of the HHE domain that contribute to the protein's iron-binding ability and/or catalase activity, and to determine whether residues on the C terminus are required for dimerization. Conserved residues D37, E42, and E95 were selected due to their proximity in the amino acid sequence to the HHE domain. Each residue was mutated to alanine using site-directed mutagenesis and the mutations were confirmed using Sanger sequencing. The E95A mutant and the C-terminal truncation mutant were expressed in Escherichia coli using the T7 expression system and purified using affinity chromatography. While wild-type Rv2633c eluted as a soluble protein, the C-terminal truncation mutant was not soluble, indicating that the C terminus may be required for Rv2633c folding. The E95A mutant eluted as a soluble protein, but may have lower iron content than wild-type Rv2633c, indicating that this glutamic acid residue could contribute to iron-binding, despite being outside the HHE domain.
Show less - Date Issued
- 2018
- Identifier
- CFH2000438, ucf:45794
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000438
- Title
- VALIDATING DRUG TARGETS THROUGH INHIBITION OF PROTEIN-PROTEIN INTERACTIONS IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
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Driscoll, Erin C, Rohde, Kyle, University of Central Florida
- Abstract / Description
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Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide...
Show moreTuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.
Show less - Date Issued
- 2017
- Identifier
- CFH2000190, ucf:46030
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000190
- Title
- DETECTION AND CHARACTERIZATION OF PATHOGENIC MYCOBACTERIA USING BINARY DEOXYRIBOZYMES.
- Creator
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Rosenkrantz, Bradley, Rohde, Kyle, University of Central Florida
- Abstract / Description
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The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world's population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest...
Show moreThe genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world's population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient's sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.
Show less - Date Issued
- 2015
- Identifier
- CFH0004758, ucf:45343
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004758
- Title
- CHARACTERIZATION OF HEMERYTHRIN-LIKE PROTEIN RV2633C.
- Creator
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Cherne, Michelle D, Self, William, University of Central Florida
- Abstract / Description
-
Hemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a �-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by...
Show moreHemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a �-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by studying the in vitro response of the recombinant protein to conditions present in the macrophage lysosome, such as reduced oxygen levels or the presence of reactive oxygen species. UV-visible spectroscopy is used to observe these changes, as the spectrum shows a characteristic peak at 330 nm that likely corresponds to the diiron cofactor in its native state. Our results show this spectrum shifts in response to hydrogen peroxide addition, showing the proposed environmental conditions can affect the active site. Bioinformatics techniques, such as the 3D modeling program SWISS-MODEL, have been used to hypothesize possible structure and function. Determining the function of Rv2633c may help explain how Mtb so readily evades the human immune system to reside in the macrophage.
Show less - Date Issued
- 2016
- Identifier
- CFH2000011, ucf:45581
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000011
- Title
- Development of Molecular Diagnostic Tools for Mycobacterium Species.
- Creator
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Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
- Abstract / Description
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This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and...
Show moreThis dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.
Show less - Date Issued
- 2017
- Identifier
- CFE0006856, ucf:51735
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006856
- Title
- Biochemical Characterization of Rv2633c from Mycobacterium tuberculosis and the Effects of Mutagenesis on Iron Binding.
- Creator
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Strickland, Kyle, Self, William, Rohde, Kyle, Davidson, Victor, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic...
Show moreMycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic conditions. Mtb employs the use of catalases, nitric oxide reductase, superoxide dismutase, and siderophores to aid in survival. These functions have also been found in a novel group of non-heme diiron binding proteins called hemerythrin-like proteins.The gene Rv2633c encodes a protein with the hemerythrin-like domain and has been shown to be upregulated under acidic or nutrient deficient conditions which coincides with Mtb infection of a macrophage. It has also been shown to be regulated by PhoP, Whib3, and DosR. In this work we expressed the wild type protein and several mutants heterologously in E. coli. The purified proteins were studied via UV-visible spectroscopic analysis, native polyacrylamide gel electrophoresis (native-PAGE) and analyzed for iron content.Our refined expression and purification protocol led to a significant increase in soluble protein with a di-iron cofactor. We found that mutagenesis of 11th amino acid, a histidine, led to the absence of the diiron co-factor. Reduction and autoxidation of protein was also achieved and characterized through UV-visible absorption. Native-PAGE gel analysis indicated only the dimeric form contained iron. This research is the first to produce large quantities of soluble iron laden protein, demonstrate that Rv2663c is capable of both reduction and autoxidation, and show it does not bind oxygen in a functional capacity. This information will enable future studies in protein crystallization, ligand interaction and in vivo studies.
Show less - Date Issued
- 2019
- Identifier
- CFE0007729, ucf:52456
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007729
- Title
- USING ANTENNA TILE-ASSISTED SUBSTRATE DELIVERY TO IMPROVE THE DETECTION LIMITS OF DEOXYRIBOZYME BIOSENSORS.
- Creator
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Cox, Amanda, Kolpashchikov, Dmitry, University of Central Florida
- Abstract / Description
-
One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion-controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor...
Show moreOne common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion-controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of Dz 10-23. The activated Dz 10-23 cleaves the fluorophore and quencher-labeled DNA-RNA substrate which separates the fluorophore from the quencher thus producing high fluorescent signal. This system uses a Dz 10-23 biosensor strand associated to a DNA antenna tile, which captures the fluorogenic substrate and channels it to the reaction center where the Dz 10-23 cleaves the substrate. DNA antenna tile captures fluorogenic substrate and delivers it to the activated Dz 10-23 core. This allows for lower levels of analyte to be detected without compromising the specificity of the biosensor. The results of this experiment demonstrated that using DNA antenna, we can create a synthetic environment around the Dz 10-23 biosensor to increase its efficiency and allow for lower levels of analyte to be detected without using amplification techniques like PCR.
Show less - Date Issued
- 2015
- Identifier
- CFH0004887, ucf:45432
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004887
- Title
- MYCOBACTERIUM TUBERCULOSIS REGULATION OF EFFLUX PUMP TAP BY TRANSCRIPTIONAL ACTIVATOR WHIB7.
- Creator
-
Pollock, Aaron, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a debilitating disease that affects the health of millions annually. Understanding its ability to persist within host and resist eradication by antibiotics is of utmost importance in the effort to develop new interventions. This study will focus on the transcriptional activator WhiB7 and its regulation of the multidrug Tap efflux pump encoded by Rv1258c. WhiB7 is thought to respond to redox stress induced by antibiotics and a...
Show moreTuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a debilitating disease that affects the health of millions annually. Understanding its ability to persist within host and resist eradication by antibiotics is of utmost importance in the effort to develop new interventions. This study will focus on the transcriptional activator WhiB7 and its regulation of the multidrug Tap efflux pump encoded by Rv1258c. WhiB7 is thought to respond to redox stress induced by antibiotics and a variety of in vivo stresses by activating multiple genes including Rv1258c. Much remains to be determined regarding the role of WhiB7 and downstream genes in Mtb virulence and drug resistance. We will create a tool for studying WhiB7-mediated gene regulation by engineering a strain of the nonpathogenic bacterium Msm expressing the mCherry fluorescent protein controlled by the Rv1258c promoter. Knocking out the native WhiB7 gene in Msm via homologous recombination will allow clear introduction of wild type and mutant versions of Mtb WhiB7. Changes in the fluorescent activity of Rv1258c promoter fusion to mCherry will indicate the effects of WhiB7 mutagenesis. Secondly, we can also use this system to confirm additional genes identified by microarray analysis that are potentially regulated by WhiB7. This will be done by cloning other promoters in front of mCherry in the Msm strain containing wild-type Mtb WhiB7. Understanding WhiB7's role in Mycobacterium tuberculosis macrophage survival and antibiotic resistance may provide new strategies for developing drugs that can lead to a cure.
Show less - Date Issued
- 2014
- Identifier
- CFH0004654, ucf:45260
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004654
- Title
- UNDERSTANDING THE ROLE OF A HEMERYTHRIN-LIKE PROTEIN IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
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Herndon, Caitlyn, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
According to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of...
Show moreAccording to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of the lung. Preliminary microarray data has shown that a TB gene known as Rv2633c is induced upon macrophage invasion. Bioinformatic analysis of Rv2633c coding sequence shows the product of Rv2633c has homology with hemerythrin-like proteins. Hemerythrins are a class of proteins commonly used to bind oxygen and sense nitric oxide and iron, leading us to hypothesize a role for Rv2633c in surviving hypoxic or nitrosative stress encountered within macrophages and granulomas. My first aim will be to generate a reporter strain of Mycobacterium smegmatis (Msm) expressing the mCherry fluorescent protein driven by the Rv2633c promoter. This tool will allow us to determine the stress conditions (i.e. hypoxia, nitric oxide treatment, acid pH) that activate expression of this gene by measuring the change in fluorescence. Linking the regulation of Rv2633c to specific environmental cues relevant to infections in vivo will provide insight into the role of this unique protein. Secondly, a knockout mutant of Rv2633c in the attenuated M. bovis BCG will be constructed and characterized to determine the importance and function of this protein during TB infections.
Show less - Date Issued
- 2014
- Identifier
- CFH0004647, ucf:45291
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004647
- Title
- Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts.
- Creator
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Lloyd, Bethany, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
- Abstract / Description
-
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment...
Show moreTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue(&)#180;rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes.LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development. ?
Show less - Date Issued
- 2012
- Identifier
- CFE0004502, ucf:49289
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004502
- Title
- Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients.
- Creator
-
Alcedo, Karel, Naser, Saleh, Cheng, Zixi, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three...
Show moreCrohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs (-) Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
Show less - Date Issued
- 2015
- Identifier
- CFE0005917, ucf:50831
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005917
- Title
- Dubious role of Mycobacterium paratuberculosis in pathogenesis of Type I diabetes.
- Creator
-
Thanigachalam, Saisathya, Naser, Saleh, Singla, Dinender, Siddiqi, Shadab, Jewett, Travis, University of Central Florida
- Abstract / Description
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INTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto...
Show moreINTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto destruction of pancreatic beta cells leading to insufficient insulin production and the development of TIDM, following exposure to MAP. METHODOLOGY: Peptide sequences of MAP-Hsp65 and GAD65 were analyzed using BLAST and PyMOL bioinformatics tools. Cross reactivity between the two proteins were evaluated using immunoblot and ELISA. Furthermore, coded EDTA blood samples were collected from 18 subjects (12 DM and 6 controls) and investigated for the presence or exposure to MAP. Peripheral leukocytes were investigated for harboring viable MAP using long-term culture followed by nested PCR. Clinical plasma samples were used for measurement of anti-MAP IgG titer as well as glucose and Insulin concentrations. Moreover, coded bovine sera from 100 cattle (50 MAP infected and 50 healthy) were investigated for possible correlation between MAP infection and plasma levels of glucose and insulin. RESULT: Peptide BLAST analysis revealed a 44% identity between MAP Hsp65 and GAD65 proteins with 75% positive identities in a 16 amino acid region. PyMOL 3-D structural analyses identified a shared epitope region within the 16 amino acid motif which is known to be an antigenic site on GAD65 antigen. MAP DNA and anti-MAP IgG were detected in the blood of TD8, a TIDM subject. Strong cross reactivity was observed between plasma from TD8 and MAP Hsp65 in proteins samples from M. tuberculosis, and E. coli recombinant clone expressing MAP Hsp65. A weak cross reactivity was also observed between rat pancreatic tissue homogenate and rabbit anti-MAP IgG. Long term culture of leukocytes from 18 blood samples resulted in the detection of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects whereas anti-MAP IgG were detected in 5/10 (50%) TIDM samples compared to 3/8 (37.5 %) controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle (P(<)0.0001). CONCLUSION: We identified and confirmed a shared epitope region between MAP Hsp65 and human pancreatic GAD65. The shared epitope is a known antigenic binding site. Although MAP DNA was detected in both TIDM and control subjects, a strong correlation was found between anti-MAP IgG titer and MAP-positive culture in clinical samples, regardless of diagnosis. The correlation between MAP infection and insulin level in cattle is significant. Overall the result is intriguing and requires further investigation of MAP in well-characterized clinical samples.
Show less - Date Issued
- 2012
- Identifier
- CFE0004608, ucf:49924
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004608
- Title
- MOLECULAR TYPING OF MYCOBACTERIAL ISOLATES CULTURED FROM THE TISSUE OF INFLAMMATORY BOWEL DISEASE (CROHN'S DISEASE) PATIENTS.
- Creator
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Adams, Leanne M, Naser, Saleh, University of Central Florida
- Abstract / Description
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The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900...
Show moreThe role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.
Show less - Date Issued
- 2004
- Identifier
- CFE0000031, ucf:46125
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000031
- Title
- DETECTION OF DRUG-RESISTANCE CONFERRING SINGLE NUCLEOTIDE POLYMORPHISMS IN MYCOBACTERIUM TUBERCULOSIS USING BINARY DNAZYMES.
- Creator
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Addario, Marina, Rohde, Kyle, University of Central Florida
- Abstract / Description
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Mycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains...
Show moreMycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains efficiently therefore more effective point-of-care (POC) diagnostics and drug susceptibility testing (DST) are urgently needed to detect drug resistance and facilitate prompt, appropriate treatment plans. In order to detect TB and efficiently identify drug resistance, this project seeks to develop a novel diagnostic technology based on deoxyribozyme (DNAzyme) sensors. The overall goal of this project is to create an assay which combines Polymerase Chain Reaction (PCR) and DNAzymes to identify drug resistance conferring Single Nucleotide Polymorphisms (SNPs). To safely test the ability of DNAzyme sensors to detect SNPs indicative of multi-drug resistant TB, we have constructed a panel of drug resistant (drugR) nonpathogenic M. bovis BCG. We have designed a multiplex PCR that amplifies 6 chromosomal regions of the genome necessary for the species specific detection of TB and determination of a drug susceptibility profile based on the presence of SNPs. To improve the sensitivity and selectivity of the detection and DST of Mtb, we have designed and optimized DNAzyme sensor assays combined with multiplex PCR analytes that will enable the rapid, POC detection of drug resistance. This work aims to develop novel tools for the prompt and specific diagnosis of TB allowing for the implementation of an iv effective treatment regimen that will ultimately lessen transmission and control the emerging global threat of MDR and XDR-TB.
Show less - Date Issued
- 2015
- Identifier
- CFH0004844, ucf:45435
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004844
- Title
- DEVELOPMENT OF NOVEL FLUORESCENT TOOLS FOR INVESTIGATING VIRULENCE FACTORS AND DRUG SUSCEPTIBILITY IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
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Wilburn, Kaley, Rohde, Kyle, University of Central Florida
- Abstract / Description
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Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for...
Show moreMycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for so long are still poorly understood at the molecular level. This lack of knowledge contributes to ongoing challenges to curing TB. Although drugs capable of killing Mtb exist, even strains that are susceptible to these drugs remain so difficult to treat that stringent six- to nine-month courses of four-drug cocktails are required. Practical difficulties in administering full treatments and patient noncompliance have contributed to a rise in drug-resistant TB cases globally. To combat this increasing world health problem, new antibiotic treatments that kill Mtb and drug-resistant Mtb more effectively via new mechanisms of action are necessary. Discovering these antibiotics expediently requires that innovative Mtb-specific drug-screening assays are developed. An ideal and innovative TB drug screening method would target validated protein-protein interactions (PPI) essential to Mtb's pathogenesis and would be performed on whole Mtb cells under relevant in vivo-like conditions. This project focused on engineering several tools relevant to creating an ideal TB drug screen. A protein fragment complementation assay capable of studying PPI of the TB gyrase complex was created, and this assay was assessed for future HTS applications. To streamline the readout, this assay was re-engineered to include green fluorescent protein. Modifications to the red fluorescent protein mCherry, including the creation of a large Stokes shift mutant mCherry and an mCherry bimolecular fluorescence complementation assay, were also engineered and investigated.
Show less - Date Issued
- 2015
- Identifier
- CFH0004843, ucf:45473
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004843
- Title
- Role of Single Nucleotide Polymorphisms (SNPs) in PTPN2/22 and Mycobacterium avium subspecies paratuberculosis (MAP) in Rheumatoid Arthritis and Crohn's Disease.
- Creator
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Sharp, Robert, Naser, Saleh, Parks, Griffith, Roy, Herve, Singla, Dinender, University of Central Florida
- Abstract / Description
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Both genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs...
Show moreBoth genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs effect on PTPN2/22 and IFN-y expression was determined using RT-PCR. Blood samples were analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. T-cell proliferation and response to phytohematoagglutonin (PHA) mitogen and MAP cell lysate were determined by BrdU proliferation assay. Out of 9 SNPs, SNP alleles of PTPN2:rs478582 occurred in 79% RA compared to 60% control (p-values ? 0.05). SNP alleles of PTPN22:rs2476601 occurred in 29% RA compared to 6% control (p-values ? 0.05). For the haplotype combination of PTPN2:rs478582/PTPN22rs2476601, 21.4% RA had both SNPs (C-A) compared to 2.4% control (p-values ? 0.05). PTPN2/22 expression in RA was decreased by an average of 1.2 fold. PTPN2:rs478582 upregulated IFN-y in RA by an average of 1.5 fold. Combined PTPN2:rs478582/PTPN22:rs2476601 increased T-cell proliferation by an average of 2.7 fold when treated with PHA. MAP DNA was detected in 34% RA compared to 8% controls (p-values ? 0.05), where samples with PTPN2:rs478582 and/or PTPN22:rs2476601 were more MAP positive. PTPN2:rs478582/PTPN22:rs2476601 together with MAP infection significantly increased T-cell response and IFN-y expression in RA samples. The same experimental approach was followed on blood samples from CD patients. Both PTPN2:rs478582/PTPN22:rs2476601 affected PTPN2/22 and IFN-y expression along with T-cell proliferation significantly more than in RA. MAP DNA was detected in 64% of CD. This is the first study to report the correlation between SNPs in PTPN2/22, IFN-y expression and MAP in autoimmune disease.
Show less - Date Issued
- 2018
- Identifier
- CFE0007371, ucf:52094
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007371
- Title
- Downregulation in IFNGR1 increases suspectiblity to Mycobacterium avium subspecies paratuberculosis infection in Crohn's disease.
- Creator
-
Htun, Zin Mar, Naser, Saleh, Andl, Claudia, Tigno-Aranjuez, Justine, University of Central Florida
- Abstract / Description
-
BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in...
Show moreBACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in association with IFNGR1 mutation in CD patients have not been investigated.METHODS: In this study, we investigated blood samples of 79 human subjects for MAP infection in association with IFNGR1 gene dysfunction. Samples were divided into 22 CD, 6 Ulcerative colitis (UC), 32 normal healthy and 19 non-inflammatory bowel disease (NIBD). Five variants of IFNGR1 single nucleotide polymorphisms (SNP) were investigated using Taqman Genotyping assay, then IFNGR1 expression measured by RT-PCR and serum IFNGR1 and ?-IFN levels were measured using ELISA. MAP infection was detected using nested PCR. RESULTS: Among 28 IBD patients, 4/6 (66.67%) of UC and 18/22 (81.82%) of CD are tested positive for at least one SNP homozygous minor form compared to 21.88% and 47.37%% in 32 healthy and 19 NIBD (P (<)0.05). IFNGR1 gene expression was downregulated 1.4-fold in IBD patients (P =0.07) and 1.7-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients (P=0.06). Serum IFNGR1 protein levels were downregulated 1.53-fold in IBD patients compared to normal, and 1.4-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients. MAP infection is more common in rs2234711 SNP positive patients (5/7 =71.42%) (P(<)0.05). Serum ?-IFN levels were not elevated in both groups.CONCLUSION: IFNGR1 SNP's, MAP infection and IFNGR1 downregulation were found in higher incidence in IBD, suggesting role of IFNGR1 in susceptibility of MAP infection in IBD patients.
Show less - Date Issued
- 2017
- Identifier
- CFE0007121, ucf:51951
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007121
- Title
- DEVELOPMENT OF A FLUORESCENT DRUG SCREENING PLATFORM FOR INHIBITORS OF MYCOBACTERIUM TUBERCULOSIS PROTEIN-PROTEIN INTERACTIONS.
- Creator
-
Versfeld, Zina, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective...
Show moreTuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective against MDR-TB. In order to discover compounds that target under-exploited pathways that may be essential only in vivo, the proposed screening platform will use a novel approach to drug discovery by blocking essential protein-protein interactions (PPI). In Mtb, PPI can be monitored by mycobacterial protein fragment complementation (M-PFC). This project will re-engineer the M-PFC assay to include the red fluorescent mCherry reporter for increased efficiency and sensitivity in high-throughput screening applications. To optimize the mCherry assay, we have developed fluorescent M-PFC reporter strains to monitor distinct PPI required for Mtb virulence: homodimerization of the dormancy regulator DosR. A drug screen will then identify novel compounds that inhibit this essential PPI. The screen will involve positional-scanning combinatorial synthetic libraries, which are made up of chemical compounds with varying side chains. This work will develop novel tools for TB drug discovery that could identify new treatments for the emerging world threat of MDR-TB.
Show less - Date Issued
- 2015
- Identifier
- CFH0004785, ucf:45369
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004785
