The de novo biosynthesis of biotin is required for the optimal growth of Salmonella enterica serovar Typhimurium in the intracellular environment
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
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
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