Elucidation of the roles of the carbonic anhydrase enzymes, CanA & CanB, in the physiology of Mycobacterium smegmatis
- Authors: Jackson, Gabriella Teresa
- Date: 2024-04-04
- Subjects: Mycobacterium tuberculosis , Mycobacterium smegmatis , Carbonic anhydrase , CRISPR , Homologous recombination , Drug targeting
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/435299 , vital:73145
- Description: The bacterial pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and one of the leading infectious causes of death globally. The success of Mtb as a pathogen depends on its ability to detect and respond to a variety of physical and chemical stresses it encounters during infection of its human host. These environmental stresses include shifts in temperature, oxygen concentration, osmolarity and nutrient availability. Mtb is, in addition, exposed to changes in pH and CO2 concentration in the intracellular and extracellular environments it inhabits, which the bacterium has to adapt to in order to ensure its growth, survival and/or persistence during infection. Carbonic anhydrases (CAs) are a widely distributed family of enzymes that catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) in the reaction: CO2 + H2O ⇄ HCO3− + H+. In microbes, CA activity is important for the activity of enzymes involved in carbon fixation as well as for maintaining pH homeostasis. Mtb is known to express three CAs, encoded by the Rv3588c, Rv1284 and Rv3273 genes (canA, canB and canC, respectively). The role(s) of these CA enzymes in the physiology of Mtb and other mycobacterial species, such as Mycobacterium smegmatis (Msm), has not been elucidated to date. To gain insights into the function of the CanA and CanB enzymes in mycobacterial species, we generated both canA and canB knockdown (KD) and knockout (KO) mutants in the fast-growing mycobacterial species, Msm, and analysed their growth phenotypes under several growth conditions where CA activity is known to be required. Notably, Msm lacks the CanC homologue, which makes it an ideal surrogate to focus on CanA and CanB. The Msm canA KD mutant was found to display a growth defect following anhydrotetracycline (ATc)-mediated gene silencing at atmospheric (low) CO2 concentrations [~0.035% CO2 (v/v)]. The growth defect could be rescued by incubating cells at physiological (high) CO2 concentrations [~5% CO2 (v/v)] or by supplementing the growth media with either HCO3− or the metabolic end-products of certain HCO3−-dependent-carboxylase enzymes at low CO2 concentrations. The ability of these compounds to rescue the growth of the canA KD mutants was, however, dependent on the extent of ATc-mediated gene silencing, suggesting that the canA gene is required for Msm growth at both low and high CO2 concentrations. This was confirmed by our findings that canA could only be genetically inactivated when a second copy of the gene was provided on the chromosome in trans, regardless of the CO2 concentration used. In contrast to our observations for canA, no differences in the growth phenotypes of the Msm wild type (WT) and canB KD or knockout (KO) mutant strains were observed following silencing or inactivation of the canB gene at either low or high CO2 concentrations or different pH values. These observations suggest that, in contrast to canA, the canB gene is dispensable for the growth of Msm under standard laboratory growth conditions. The canB KO mutant strain, nevertheless, displayed a slight decrease in its ability to form biofilms when compared to the WT strain, which could be restored by genetic complementation. CanB activity may, therefore, be required to promote bacterial growth and/or survival under biofilm conditions where CO2 diffusion into cells is limited, a phenomenon that has recently been observed in other microbes. Further studies are required to confirm the role of CanB in biofilm formation and to determine how the different CA enzymes cooperate to promote the growth and survival of mycobacterial species in the various environments they are known to inhabit. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2024
- Full Text:
- Date Issued: 2024-04-04
- Authors: Jackson, Gabriella Teresa
- Date: 2024-04-04
- Subjects: Mycobacterium tuberculosis , Mycobacterium smegmatis , Carbonic anhydrase , CRISPR , Homologous recombination , Drug targeting
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/435299 , vital:73145
- Description: The bacterial pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and one of the leading infectious causes of death globally. The success of Mtb as a pathogen depends on its ability to detect and respond to a variety of physical and chemical stresses it encounters during infection of its human host. These environmental stresses include shifts in temperature, oxygen concentration, osmolarity and nutrient availability. Mtb is, in addition, exposed to changes in pH and CO2 concentration in the intracellular and extracellular environments it inhabits, which the bacterium has to adapt to in order to ensure its growth, survival and/or persistence during infection. Carbonic anhydrases (CAs) are a widely distributed family of enzymes that catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) in the reaction: CO2 + H2O ⇄ HCO3− + H+. In microbes, CA activity is important for the activity of enzymes involved in carbon fixation as well as for maintaining pH homeostasis. Mtb is known to express three CAs, encoded by the Rv3588c, Rv1284 and Rv3273 genes (canA, canB and canC, respectively). The role(s) of these CA enzymes in the physiology of Mtb and other mycobacterial species, such as Mycobacterium smegmatis (Msm), has not been elucidated to date. To gain insights into the function of the CanA and CanB enzymes in mycobacterial species, we generated both canA and canB knockdown (KD) and knockout (KO) mutants in the fast-growing mycobacterial species, Msm, and analysed their growth phenotypes under several growth conditions where CA activity is known to be required. Notably, Msm lacks the CanC homologue, which makes it an ideal surrogate to focus on CanA and CanB. The Msm canA KD mutant was found to display a growth defect following anhydrotetracycline (ATc)-mediated gene silencing at atmospheric (low) CO2 concentrations [~0.035% CO2 (v/v)]. The growth defect could be rescued by incubating cells at physiological (high) CO2 concentrations [~5% CO2 (v/v)] or by supplementing the growth media with either HCO3− or the metabolic end-products of certain HCO3−-dependent-carboxylase enzymes at low CO2 concentrations. The ability of these compounds to rescue the growth of the canA KD mutants was, however, dependent on the extent of ATc-mediated gene silencing, suggesting that the canA gene is required for Msm growth at both low and high CO2 concentrations. This was confirmed by our findings that canA could only be genetically inactivated when a second copy of the gene was provided on the chromosome in trans, regardless of the CO2 concentration used. In contrast to our observations for canA, no differences in the growth phenotypes of the Msm wild type (WT) and canB KD or knockout (KO) mutant strains were observed following silencing or inactivation of the canB gene at either low or high CO2 concentrations or different pH values. These observations suggest that, in contrast to canA, the canB gene is dispensable for the growth of Msm under standard laboratory growth conditions. The canB KO mutant strain, nevertheless, displayed a slight decrease in its ability to form biofilms when compared to the WT strain, which could be restored by genetic complementation. CanB activity may, therefore, be required to promote bacterial growth and/or survival under biofilm conditions where CO2 diffusion into cells is limited, a phenomenon that has recently been observed in other microbes. Further studies are required to confirm the role of CanB in biofilm formation and to determine how the different CA enzymes cooperate to promote the growth and survival of mycobacterial species in the various environments they are known to inhabit. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2024
- Full Text:
- Date Issued: 2024-04-04
The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
Investigating cannabinoids and endocannabinoid receptors as drug targets for pain and inflammation
- Authors: Marwarwa, Sinobomi Zamachi
- Date: 2020
- Subjects: Cannabinoids , Cannabinoids Receptors , Inflammation Alternative treatment , Pain Alternative treatment , Drug targeting
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/164468 , vital:41121
- Description: Cannabinoids and the endocannabinoid system have been studied in the past decades but have yet to be fully understood. An insight into interactions that occur between cannabinoid compounds and their receptors is important for understanding the cannabinoids and the endocannabinoid system. Cannabinoids are natural products found in some cannabis plants, and they have similar effects to endocannabinoids, which are chemicals in the body that are involved many aspects of health from appetite, memory, and movement to pain, inflammation and response to cancer. Cannabinoids have a high impact on the treatment of pain and inflammation, they show different antinociceptive mechanisms to existing drugs like opioids, also, they have antimigraine properties better than those achieved by aspirin. The CB1 and CB2 human receptors have been the most studied cannabinoid receptors. In this project, we used a combination of mass-spectrometry to generate plausible chemical fragments and computational techniques to assess the binding of these fragments to these two main CB receptors. CB1 was adapted from the protein data bank (PBD), file 5U09 and the CB2 model was predicted using the hierarchical protocol I-TASSER, starting from the amino acid sequence in UniProt (P34972 CNR2_HUMAN). The proposed active site for CB1 was reported in a publication accompanying the 5U09 PDB model, which was originally generated with a pre-existing ligand in the active site. However, CB2 had to be built from a homology model and the active site determined using a combination of I-TASSER, Maestro, and CASTp the more favourable binding energies were determined by CASTp, leading to the use of the CASTp coordinates as default for docking in the CB2 human receptor. The molecular docking of cannabinoids THC, CBD, CBDV, CBG and CBN on both the CB1 and CB2 proteins was performed to identify the amino acids that interact with these compounds at their active sites. This would provide a guide to a future fragment-based drug discovery (FBDD) synthesis project. The docking in this work showed adequate accuracy with binding energies between -8.23 kcal/mol and -9.97 kcal/mol for CB1 and between -6.78 kcal/mol and -7.74 kcal/mol for CB2. An observation made was that binding energies of the CB1 human receptor docking were higher than those of the CB2 human receptor, which could support the widely held belief that CB1 is more important in cannabinoid interactions. The cannabinoids were then subjected to collision-induced dissociation to produce fragment structures predicted in chapter 2. These hypothetical fragments were docked in the CB1 and CB2 human receptor, the general trend again being the binding energies for the CB1 receptor was again around 10% higher than those of the CB2 receptor. As expected, larger fragments tended to have better binding, with the fragment proposed from m/z 259 with binding energies -9.62 kcal/mol in CB1 and -6.26 kcal/mol. Those fragments with significant lipophilic side chains or some aromatic moiety also showed good binding or around -6.00 kcal/mol, similar to the intact cannabinoids. In our case, this fragment was proposed from m/z 223 with binding energies -7.71 kcal/mol in CB1 and -6.5 kcal/mol in CB2. The results from the fragment dockings were favourable in that they have binding affinities lower than -6.0 kcal/mol which is good enough for the structures to be leads in the creation of fragment libraries. The docking was performed with Autodock 1.5.6 and data visualization with a discovery studio. , Thesis (MSc) -- Faculty of Science, Chemistry, 2020
- Full Text:
- Date Issued: 2020
- Authors: Marwarwa, Sinobomi Zamachi
- Date: 2020
- Subjects: Cannabinoids , Cannabinoids Receptors , Inflammation Alternative treatment , Pain Alternative treatment , Drug targeting
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/164468 , vital:41121
- Description: Cannabinoids and the endocannabinoid system have been studied in the past decades but have yet to be fully understood. An insight into interactions that occur between cannabinoid compounds and their receptors is important for understanding the cannabinoids and the endocannabinoid system. Cannabinoids are natural products found in some cannabis plants, and they have similar effects to endocannabinoids, which are chemicals in the body that are involved many aspects of health from appetite, memory, and movement to pain, inflammation and response to cancer. Cannabinoids have a high impact on the treatment of pain and inflammation, they show different antinociceptive mechanisms to existing drugs like opioids, also, they have antimigraine properties better than those achieved by aspirin. The CB1 and CB2 human receptors have been the most studied cannabinoid receptors. In this project, we used a combination of mass-spectrometry to generate plausible chemical fragments and computational techniques to assess the binding of these fragments to these two main CB receptors. CB1 was adapted from the protein data bank (PBD), file 5U09 and the CB2 model was predicted using the hierarchical protocol I-TASSER, starting from the amino acid sequence in UniProt (P34972 CNR2_HUMAN). The proposed active site for CB1 was reported in a publication accompanying the 5U09 PDB model, which was originally generated with a pre-existing ligand in the active site. However, CB2 had to be built from a homology model and the active site determined using a combination of I-TASSER, Maestro, and CASTp the more favourable binding energies were determined by CASTp, leading to the use of the CASTp coordinates as default for docking in the CB2 human receptor. The molecular docking of cannabinoids THC, CBD, CBDV, CBG and CBN on both the CB1 and CB2 proteins was performed to identify the amino acids that interact with these compounds at their active sites. This would provide a guide to a future fragment-based drug discovery (FBDD) synthesis project. The docking in this work showed adequate accuracy with binding energies between -8.23 kcal/mol and -9.97 kcal/mol for CB1 and between -6.78 kcal/mol and -7.74 kcal/mol for CB2. An observation made was that binding energies of the CB1 human receptor docking were higher than those of the CB2 human receptor, which could support the widely held belief that CB1 is more important in cannabinoid interactions. The cannabinoids were then subjected to collision-induced dissociation to produce fragment structures predicted in chapter 2. These hypothetical fragments were docked in the CB1 and CB2 human receptor, the general trend again being the binding energies for the CB1 receptor was again around 10% higher than those of the CB2 receptor. As expected, larger fragments tended to have better binding, with the fragment proposed from m/z 259 with binding energies -9.62 kcal/mol in CB1 and -6.26 kcal/mol. Those fragments with significant lipophilic side chains or some aromatic moiety also showed good binding or around -6.00 kcal/mol, similar to the intact cannabinoids. In our case, this fragment was proposed from m/z 223 with binding energies -7.71 kcal/mol in CB1 and -6.5 kcal/mol in CB2. The results from the fragment dockings were favourable in that they have binding affinities lower than -6.0 kcal/mol which is good enough for the structures to be leads in the creation of fragment libraries. The docking was performed with Autodock 1.5.6 and data visualization with a discovery studio. , Thesis (MSc) -- Faculty of Science, Chemistry, 2020
- Full Text:
- Date Issued: 2020
- «
- ‹
- 1
- ›
- »