Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
Targeting allosteric sites of Escherichia coli heat shock protein 70 for antibiotic development
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
Applicability of putative probiotic bacteria to replace antibiotics as growth promoters in commercial and indigenous piglets
- Authors: Dlamini, Ziyanda Confidence
- Date: 2017
- Subjects: Antibiotics , Dietary supplements , Piglets
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/11428 , vital:39071
- Description: Antibiotic use in pig production contributes to development of antimicrobial resistance in food animals and risk of antibiotic residues in animal food products. The use of probiotics appears to be the potential alternative to antibiotics. This work is designed to validate the applicability of administered direct-fed putative probiotic strains of Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4, Lactobacillus salivarius ZJ614 and Streptococcus salivarius NBCR 13956 on growth performance, hematological parameters, and antibody stimulation in weaned commercial and indigenous pigs. Forty-five weaned piglets were divided into five treatment experimental groups as follows: Diet with: antibiotic (PC), No antibiotic and no probiotic (NC), Probiotic (P1) (P2) combination of probiotics (P3). Piglets’ performance were monitored during the trial. Faecal and ileum samples were collected for microbial count analysis. Blood samples were collected from the sacrificed piglets at the end of the trial, for the hematological and biochemical analysis and probiotics ability to stimulate immunoglobulin G (IgG). Four probiotic strains were tested for cell surface hydrophobicity against xylene, chloroform and ethyl acetate. The strains were also tested for auto-aggregation and co-aggregation abilities. Enterococcus faecalis ATCC 29212 was used as a control pathogen in the co-aggregation ability test. The probiotic strains were also screened for presence of virulence and resistance genes. DNA was extracted from all the four probiotic strains using ZR Fungal/Bacterial DNA MiniPepTM (Zymo Research, USA) following manufacturer’s instructions. The DNA samples were later amplified in PCR reactions with specific primers to detect virulence genes of adhesion collagen protein (ace), aggregation substances (agg and asa); antibiotic resistance genes of: Vancomycin vanA, Vancomycin vanC1 and Vancomycin vanC2/3. The data was analyzed by one-way ANOVA using SAS statistics software (SAS 9.3) (2003). Results from the study revealed that supplementation of probiotics had no effect on feed intake (FI) in all of the experimental groups. However, supplementation of probiotics in P3 treatment resulted in greater average daily gain (ADG) and improved feed conversion ratio (FCR) of weaned piglets (p < 0.05). Microbial count of fecal samples from all the treatment groups did not differ while ileum samples had lower enteric bacteria in P3 group as compared to other treatments. Concentration of albumin, globulin, neutrophils and basophils were high in NC treatment compared to other treatments. (p < 0.05). IgG concentration was highest in P3 compared to other treatment (p < 0.05). All probiotics strains showed high hydrophobicity to all solvents used in the test. Cell Surface Hydrophobic was highest in xylene (78percent – 84percent), followed by chloroform (68percent – 75percent) and lowest in ethyl acetate (52percent - 60percent). All of the test strains showed high auto-aggregation ability after 4 hr of incubation. L .reuteri VB4 exhibited highest auto-aggregation ability of 70percent, while the least auto-aggregation ability of 60percent was observed for L. salivarius ZJ614. In the co-aggregation assays, all probiotic bacterial strains exhibited a strong co-aggregation of 45percent to 56percent after 4 hr of incubation at 37 °C. L. reuteri VB4 exhibited highest co-aggregation ability while L. salivarius ZJ614 exhibited the lowest co-aggregation ability. No presence of virulence genes ace and asa was detected in all of the putative probiotic strains, but agg virulence gene was detected in L. reuteri VB4 strains. L. reuteri VB4 and S. salivarius NBRC13956 had resistance genes vanC 2/3 and vanC1, respectively. Overall, the results from this study, suggest that the candidate probiotics strains have enhanced the growth and measured blood parameters of the weaned piglets used in these experiments, and also, the probiotic strains have the abilities to initiate the stimulation of IgG. Probiotics could be used as a novel alternative to antibiotics in the animal nutrition and production, with the abilities to confer health benefits and promote growth. The outcome of the research advocates that these probiotics will be beneficial to pig production, and might outcompete the current antibiotics in use as growth promoters. One of the major advantages of probiotics to pig industry is the production of antibiotic free and good quality pork meat to local and international consumers, thus, enhancing South African Pork industry.
- Full Text:
- Date Issued: 2017
- Authors: Dlamini, Ziyanda Confidence
- Date: 2017
- Subjects: Antibiotics , Dietary supplements , Piglets
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/11428 , vital:39071
- Description: Antibiotic use in pig production contributes to development of antimicrobial resistance in food animals and risk of antibiotic residues in animal food products. The use of probiotics appears to be the potential alternative to antibiotics. This work is designed to validate the applicability of administered direct-fed putative probiotic strains of Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4, Lactobacillus salivarius ZJ614 and Streptococcus salivarius NBCR 13956 on growth performance, hematological parameters, and antibody stimulation in weaned commercial and indigenous pigs. Forty-five weaned piglets were divided into five treatment experimental groups as follows: Diet with: antibiotic (PC), No antibiotic and no probiotic (NC), Probiotic (P1) (P2) combination of probiotics (P3). Piglets’ performance were monitored during the trial. Faecal and ileum samples were collected for microbial count analysis. Blood samples were collected from the sacrificed piglets at the end of the trial, for the hematological and biochemical analysis and probiotics ability to stimulate immunoglobulin G (IgG). Four probiotic strains were tested for cell surface hydrophobicity against xylene, chloroform and ethyl acetate. The strains were also tested for auto-aggregation and co-aggregation abilities. Enterococcus faecalis ATCC 29212 was used as a control pathogen in the co-aggregation ability test. The probiotic strains were also screened for presence of virulence and resistance genes. DNA was extracted from all the four probiotic strains using ZR Fungal/Bacterial DNA MiniPepTM (Zymo Research, USA) following manufacturer’s instructions. The DNA samples were later amplified in PCR reactions with specific primers to detect virulence genes of adhesion collagen protein (ace), aggregation substances (agg and asa); antibiotic resistance genes of: Vancomycin vanA, Vancomycin vanC1 and Vancomycin vanC2/3. The data was analyzed by one-way ANOVA using SAS statistics software (SAS 9.3) (2003). Results from the study revealed that supplementation of probiotics had no effect on feed intake (FI) in all of the experimental groups. However, supplementation of probiotics in P3 treatment resulted in greater average daily gain (ADG) and improved feed conversion ratio (FCR) of weaned piglets (p < 0.05). Microbial count of fecal samples from all the treatment groups did not differ while ileum samples had lower enteric bacteria in P3 group as compared to other treatments. Concentration of albumin, globulin, neutrophils and basophils were high in NC treatment compared to other treatments. (p < 0.05). IgG concentration was highest in P3 compared to other treatment (p < 0.05). All probiotics strains showed high hydrophobicity to all solvents used in the test. Cell Surface Hydrophobic was highest in xylene (78percent – 84percent), followed by chloroform (68percent – 75percent) and lowest in ethyl acetate (52percent - 60percent). All of the test strains showed high auto-aggregation ability after 4 hr of incubation. L .reuteri VB4 exhibited highest auto-aggregation ability of 70percent, while the least auto-aggregation ability of 60percent was observed for L. salivarius ZJ614. In the co-aggregation assays, all probiotic bacterial strains exhibited a strong co-aggregation of 45percent to 56percent after 4 hr of incubation at 37 °C. L. reuteri VB4 exhibited highest co-aggregation ability while L. salivarius ZJ614 exhibited the lowest co-aggregation ability. No presence of virulence genes ace and asa was detected in all of the putative probiotic strains, but agg virulence gene was detected in L. reuteri VB4 strains. L. reuteri VB4 and S. salivarius NBRC13956 had resistance genes vanC 2/3 and vanC1, respectively. Overall, the results from this study, suggest that the candidate probiotics strains have enhanced the growth and measured blood parameters of the weaned piglets used in these experiments, and also, the probiotic strains have the abilities to initiate the stimulation of IgG. Probiotics could be used as a novel alternative to antibiotics in the animal nutrition and production, with the abilities to confer health benefits and promote growth. The outcome of the research advocates that these probiotics will be beneficial to pig production, and might outcompete the current antibiotics in use as growth promoters. One of the major advantages of probiotics to pig industry is the production of antibiotic free and good quality pork meat to local and international consumers, thus, enhancing South African Pork industry.
- Full Text:
- Date Issued: 2017
Sequence and structural investigation of the nonribosomal peptide synthetases of Bacillus atrophaeus UCMB 5137(63Z)
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
The investigation of novel marine microorganisms for the production of biologically active metabolites
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
Antimicrobial resistance patterns in a Port Elizabeth hospital
- Authors: Meiring, Jillian A
- Date: 1993
- Subjects: Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4043 , http://hdl.handle.net/10962/d1004104 , Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Description: Antibiotic resistance in clinical bacterial isolates remains an ongoing problem requiring continuous monitoring to effect some form of control. Comparative studies have not been previously reported for the Eastern Cape Region, South Africa and this study was undertaken to monitor resistance patterns in clinical isolates from Provincial Hospital, Port Elizabeth. Over the three year period 1989 to 1991, 9888 susceptibility results from isolates examined in the SAIMR pathology laboratory were analysed and collated using a stand-alone computer program. Resistance patterns for a range of nineteen antibiotics were collated for isolates from various sampling points within the hospital. Results were reported as resistance patterns in individually isolated species. Levels of resistance in each species were compared to those reported from South Africa and abroad, and changing patterns of resistance were noted within the three year period at the Provincial Hospital, Port Elizabeth.
- Full Text:
- Date Issued: 1993
- Authors: Meiring, Jillian A
- Date: 1993
- Subjects: Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4043 , http://hdl.handle.net/10962/d1004104 , Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Description: Antibiotic resistance in clinical bacterial isolates remains an ongoing problem requiring continuous monitoring to effect some form of control. Comparative studies have not been previously reported for the Eastern Cape Region, South Africa and this study was undertaken to monitor resistance patterns in clinical isolates from Provincial Hospital, Port Elizabeth. Over the three year period 1989 to 1991, 9888 susceptibility results from isolates examined in the SAIMR pathology laboratory were analysed and collated using a stand-alone computer program. Resistance patterns for a range of nineteen antibiotics were collated for isolates from various sampling points within the hospital. Results were reported as resistance patterns in individually isolated species. Levels of resistance in each species were compared to those reported from South Africa and abroad, and changing patterns of resistance were noted within the three year period at the Provincial Hospital, Port Elizabeth.
- Full Text:
- Date Issued: 1993
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