Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
- Full Text:
- Date Issued: 2014
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
- Full Text:
- Date Issued: 2014
Genetic diversity and population structure of plasmodium falciparum from four epidemiological locations in Malawi
- Authors: Selemani, George Paul
- Date: 2014
- Subjects: Plasmodium falciparum , Parasites -- Molecular genetics , Infection -- Genetic aspects , Disease susceptibility -- Genetic aspects
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10359 , http://hdl.handle.net/10948/d1021026
- Description: In malaria-endemic regions, Plasmodium falciparum (P. falciparum) infection is characterized by extensive genetic/antigenic diversity. Describing this diversity provides important information about the local molecular epidemiology of infecting P. falciparum parasites. Intriguingly, one of the major obstacles to the development of an effective malaria vaccine has been the genetic polymorphisms exhibited by P. falciparum genes encoding targets of human immune system. This situation has necessitated the development of polyvalent vaccines with wide antigenic coverage that would increase the likelihood of vaccine efficacy that covers wide geographical areas of malaria endemic countries. Limited reports are available on the population genetic diversity and structure of P. falciparum in Malawi, and this is of particular concern as the country has put in place several interventions to combat the disease. The primary aim of the research project was to determine the genetic diversity and population structure of P. falciparum isolates and comparing complexity from four different epidemiological settings in Malawi using msp-2 gene polymorphisms. Samples were collected from four epidemiological locations in the north, centre and southern regions of Malawi. The diversity and genetic differentiation of P. falciparum populations were analyzed based on the highly polymorphic block 3 msp-2 gene. One hundred and twenty patient samples who presented with signs and symptoms of malaria and who had microscopically confirmed P. falciparum infection were enrolled in the study after they had satisfied the inclusion criteria. Parasite DNA was extracted from the blood spot on to filter paper and analyzed by genotyping the msp-2 gene using allele-specific nested PCR. A total of 28 msp-2 block 3 fragments, defined by the size and the allelic types were detected in the 102 patients. The length variants of the PCR product ranged from 240basepairs (bp) to 450bp for the K1/FC and 410bp to 780bp for the 3D7/IC allelic families. Isolates of the 3D7 alleles were predominant in the population (59 percent), compared to isolates of the K1/ FC27 alleles (41 percent) and for 3D7 and K1 most of the isolates were monoclonal infections. In comparisons between the sites, we observed the highest prevalence of mixed infection in Mwanza (46.7 percent) followed by Dwangwa (23.3 percent) compared to Bolero (16.7 percent) and Mitundu (16.7 percent). The difference in prevalence of mixed infections between Mwanza and the other sites was statistically significant (p=0.041). There was also a non-significant trend towards a higher mean genotype number per isolate in the children aged >5 years compared to those below 5 years of age. These data suggest differences in prevalence rates of mixed infections in different geographical/epidemiological settings in Malawi. Further studies are needed to confirm, with larger sample sizes, the observation of a non-significant trend towards higher multiclonality of infection in older children in malaria endemic areas of Malawi.
- Full Text:
- Date Issued: 2014
- Authors: Selemani, George Paul
- Date: 2014
- Subjects: Plasmodium falciparum , Parasites -- Molecular genetics , Infection -- Genetic aspects , Disease susceptibility -- Genetic aspects
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10359 , http://hdl.handle.net/10948/d1021026
- Description: In malaria-endemic regions, Plasmodium falciparum (P. falciparum) infection is characterized by extensive genetic/antigenic diversity. Describing this diversity provides important information about the local molecular epidemiology of infecting P. falciparum parasites. Intriguingly, one of the major obstacles to the development of an effective malaria vaccine has been the genetic polymorphisms exhibited by P. falciparum genes encoding targets of human immune system. This situation has necessitated the development of polyvalent vaccines with wide antigenic coverage that would increase the likelihood of vaccine efficacy that covers wide geographical areas of malaria endemic countries. Limited reports are available on the population genetic diversity and structure of P. falciparum in Malawi, and this is of particular concern as the country has put in place several interventions to combat the disease. The primary aim of the research project was to determine the genetic diversity and population structure of P. falciparum isolates and comparing complexity from four different epidemiological settings in Malawi using msp-2 gene polymorphisms. Samples were collected from four epidemiological locations in the north, centre and southern regions of Malawi. The diversity and genetic differentiation of P. falciparum populations were analyzed based on the highly polymorphic block 3 msp-2 gene. One hundred and twenty patient samples who presented with signs and symptoms of malaria and who had microscopically confirmed P. falciparum infection were enrolled in the study after they had satisfied the inclusion criteria. Parasite DNA was extracted from the blood spot on to filter paper and analyzed by genotyping the msp-2 gene using allele-specific nested PCR. A total of 28 msp-2 block 3 fragments, defined by the size and the allelic types were detected in the 102 patients. The length variants of the PCR product ranged from 240basepairs (bp) to 450bp for the K1/FC and 410bp to 780bp for the 3D7/IC allelic families. Isolates of the 3D7 alleles were predominant in the population (59 percent), compared to isolates of the K1/ FC27 alleles (41 percent) and for 3D7 and K1 most of the isolates were monoclonal infections. In comparisons between the sites, we observed the highest prevalence of mixed infection in Mwanza (46.7 percent) followed by Dwangwa (23.3 percent) compared to Bolero (16.7 percent) and Mitundu (16.7 percent). The difference in prevalence of mixed infections between Mwanza and the other sites was statistically significant (p=0.041). There was also a non-significant trend towards a higher mean genotype number per isolate in the children aged >5 years compared to those below 5 years of age. These data suggest differences in prevalence rates of mixed infections in different geographical/epidemiological settings in Malawi. Further studies are needed to confirm, with larger sample sizes, the observation of a non-significant trend towards higher multiclonality of infection in older children in malaria endemic areas of Malawi.
- Full Text:
- Date Issued: 2014
Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum
- Yao, Jia
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
The interaction of silver nanoparticles with triosephosphate isomerase from human and malarial parasite (Plasmodium falciparum) : a comparative study
- De Moor, Warren Ralph Josephus
- Authors: De Moor, Warren Ralph Josephus
- Date: 2014
- Subjects: Silver , Nanoparticles , Triose-phosphate isomerase , Plasmodium falciparum , Nanotechnology , Antimalarials , Povidone
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4169 , http://hdl.handle.net/10962/d1020895
- Description: The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.
- Full Text:
- Date Issued: 2014
- Authors: De Moor, Warren Ralph Josephus
- Date: 2014
- Subjects: Silver , Nanoparticles , Triose-phosphate isomerase , Plasmodium falciparum , Nanotechnology , Antimalarials , Povidone
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4169 , http://hdl.handle.net/10962/d1020895
- Description: The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.
- Full Text:
- Date Issued: 2014
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