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
Structural bioinformatics studies and tool development related to drug discovery
- Authors: Hatherley, Rowan
- Date: 2016
- Subjects: Structural bioinformatics , Drug development , Natural products -- Databases , Natural products -- Biotechnology , Sequence alignment (Bioinformatics) , Malaria -- Chemotherapy , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4164 , http://hdl.handle.net/10962/d1020021
- Description: This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.
- Full Text:
- Date Issued: 2016
- Authors: Hatherley, Rowan
- Date: 2016
- Subjects: Structural bioinformatics , Drug development , Natural products -- Databases , Natural products -- Biotechnology , Sequence alignment (Bioinformatics) , Malaria -- Chemotherapy , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4164 , http://hdl.handle.net/10962/d1020021
- Description: This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.
- Full Text:
- Date Issued: 2016
Towards development of a malaria diagnostic: Generation, screening and validation of novel aptamers recognising Plasmodium falciparum lactate dehydrogenase
- Authors: Frith, Kelly-Anne
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Oligonucleotides , Lactate dehydrogenase , Biochemical markers , Systematic evolution of ligands through exponential enrichment (SELEX)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142247 , vital:38062
- Description: Malaria, caused by infection with the Plasmodium parasite, is one of the leading causes of death in under-developed countries. Early detection is crucial for the effective treatment of malaria, particularly in cases where infection is due to Plasmodium falciparum. There is, therefore, an enduring need for portable, sensitive, reliable, accurate, durable, self-validating and cost-effective techniques for the rapid detection of malaria. Moreover, there is a demand to distinguish between various infectious species causing malaria. Research in the area of malarial biomarkers has identified a unique, species-specific, epitope of P. falciparum lactate dehydrogenase (PfLDH), enhancing prospects for the development of diagnostics capable of identifying the species causing malarial infection. In recent years, improvements have been made towards the development of rapid diagnostic tests for detecting malarial biomarkers. Owing to their low cost, ease of labeling, and high thermal stability (relative to antibodies), the development and synthesis of aptamers that target the malarial lactate dehydrogenase represents one of the key innovations in the field of rapid diagnostics for malaria. This study explored the generation of aptamers that specifically target P. falciparum. Two sets of aptamers with diagnostically-supportive functions were generated independently, through parallel SELEX of recombinantly-expressed, full-length Plasmodium falciparum lactate dehydrogenase (rPfLDH), and an oligopeptide comprising the P. falciparum-specific epitope on lactate dehydrogenase (LDHp). The latter offers a promising solution for generating aptamers capable of binding with high specificity to P. falciparum. In this work, an rLDH class of aptamers was generated when SELEX was performed using the full-length rPfLDH protein as the target and the LDHp class of aptamers was generated when SELEX was performed using the oligopeptide LDHp as a target. Aptamers were successfully generated through the process of SELEX (systematic evolution of ligands through exponential enrichment) following the study and application of several optimisation steps, particularly during the amplification stage of SELEX. Optimisation steps included the study of improvements in PCR conditions; role of surfactants (Triton-X), modifying the PCR clean-up protocol; and agarose gel excision. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers both reported here and previously published, confirming their importance in recognition of the target. Novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Clades of consensus sequences were identified in both the rLDH and LDHp groups of aptamers, where sequences in the rLDH clade did not show preferential binding to rPfLDH while those in the LDHp clade (particularly LDHp 3 and 18) were able to recognise and bind only LDHp. Of the 19 sequences returned from the parallel SELEX procedures for rPfLDH (11 sequences) and LDHp (8 sequences), six rPfLDH and all eight LDHp sequences underwent preliminary screening and those with low responses eliminated. Of the eight LDHp-targeting aptamer sequences, five were preliminarily shown to bind to LDHp, whereas only two rPfLDH-targeting sequences were shown to bind to the target (rLDH 4 and 7). To this small selection of rPfLDH oligonucleotide sequences, two more (rLDH 1 and 15) were chosen for further study based on their sequences, secondary and predicted tertiary conformations. Sequences chosen for further study were therefore: rLDH 1, 4, 7 and 15 in the rLDH class, and LDHp 1, 3, 11, 14 and 18 in the LDHp class. Binding properties of the aptamers towards their targets were investigated using enzyme-linked oligonucleotide assays (ELONA), fluorophore-linked oligonucleotide assays (FLONA), electromobility shift assays (EMSA), surface plasmon resonance (SPR), and GelRed dissociation assays, while applications towards aptasensors were explored using electrochemical impedance spectroscopy (EIS) and fluorescent microscopy. Some inconsistencies were seen for specific aptamer to target binding interactions using specific techniques; however, generally, binding to the targets was observed across the techniques assessed. These varied responses demonstrate the need to screen and validate aptamers using a variety of techniques and platforms not necessarily specific for the proposed application. From the aptamer binding screening studies using ELONA, the most promising aptamers generated were identified as LDHp 11, rLDH 4, rLDH 7 and rLDH 15. Aptamer rLDH 4, which was generated against rPfLDH, exhibited preferential and specific binding to the lactate dehydrogenase from P. falciparum, over the recombinantly-expressed lactate dehydrogenase from Plasmodium vivax (rPvLDH), albeit with lowered responses compared to LDHp 11 in ELONA and EMSA studies. However, in kinetic ELONA studies rLDH 4 showed binding to both rPfLDH and rPvLDH. Aptamer rLDH 7 showed high affinity for rPfLDH and rPvLDH in kinetic studies using ELONA. However, screening studies with ELONA indicates that aptamer rLDH 7 may not be suitable for diagnostic tests in serum samples given its non-specific binding to human serum albumin (HSA). Aptamer rLDH 15 exhibited species specificity for rPfLDH in screening studies using ELONA but showed affinity towards rPvLDH (albeit lower relative to its affinity for rPfLDH) in kinetic studies using ELONA. LDHp 11, generated against the PfLDH peptide, showed a clear preference for rPfLDH when compared to rPvLDH and other control proteins, in both sets of ELONA studies conducted, as well as EMSA, thus possessing a strong ability to identify the presence of Plasmodium falciparum owing to its generation against the species-specific epitope. While LDHp 1 demonstrated binding to plasmodial LDH in a flow-through system (SPR), so reiterating ELONA responses, it did not perform well in the remaining methodologies. Aptamers rLDH 1 and 15 and LDHp 3, 14 and 18 exhibited a mixed set of results throughout the target protein screening analyses and were, thus, not considered for selective binding in P. falciparum parasite bodies. In studies aimed at exploring biosensor assemblies utilising the developed aptamers, both rLDH 4 and LDHp 11, along with rLDH 7, LDHp 1 and pL1, demonstrated in situ binding to the native PfLDH in fluorescent microscopy. LDHp 11 exhibited FITC-based fluorescence equivalent to the anti-rPfLDHp IgY antibody in confocal fluorescent microscopy indicating superior binding to the native PfLDH compared to the remaining aptamers. An examination of electrochemical impedance as a platform for a biosensor assembly did not, in these studies, exhibit the required sensitivity using physiologically relevant concentrations of analyte expected for pLDH following infection with Plasmodium spp. Malstat/LDH activity was explored for application in a colorimetric aptasensor. A decrease in both rPfLDH and rPvLDH activity was observed following incubation with the tested aptamers, but rLDH 1, rLDH 7 and LDHp 14 did not exhibit similar decreases in rPvLDH activity. Aptamers rLDH 1, 4 and 7 and LDHp 11 and 14 were, therefore, not selected as candidates for LDH capture in LDH activity-based diagnostic devices for P. falciparum. The decreases in pLDH activity in the presence of aptamers could hold promise as direct or antagonistic malaria therapeutic agents. Preliminary studies on the application of aptamers as malaria therapeutic agents, while of interest, should be viewed with due caution given the challenges of aptamers reaching the intracellular native plasmodial LDH hosted within the red blood cells. In conclusion, this work has shown the ability of the LDHp 11 aptamer, generated in these studies, to selectively bind rPfLDH over rPvLDH, and to bind to the native PfLDH in fluorescent microscopy, indicating that this aptamer holds promise as a biorecognition element in malaria biosensors and other diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax. The use of a species-specific epitope of P. falciparum as a target in aptamer generation paves the way for similar such studies aimed at generating aptamers with species selectivity for other Plasmodium species.
- Full Text:
- Date Issued: 2020
- Authors: Frith, Kelly-Anne
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Oligonucleotides , Lactate dehydrogenase , Biochemical markers , Systematic evolution of ligands through exponential enrichment (SELEX)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142247 , vital:38062
- Description: Malaria, caused by infection with the Plasmodium parasite, is one of the leading causes of death in under-developed countries. Early detection is crucial for the effective treatment of malaria, particularly in cases where infection is due to Plasmodium falciparum. There is, therefore, an enduring need for portable, sensitive, reliable, accurate, durable, self-validating and cost-effective techniques for the rapid detection of malaria. Moreover, there is a demand to distinguish between various infectious species causing malaria. Research in the area of malarial biomarkers has identified a unique, species-specific, epitope of P. falciparum lactate dehydrogenase (PfLDH), enhancing prospects for the development of diagnostics capable of identifying the species causing malarial infection. In recent years, improvements have been made towards the development of rapid diagnostic tests for detecting malarial biomarkers. Owing to their low cost, ease of labeling, and high thermal stability (relative to antibodies), the development and synthesis of aptamers that target the malarial lactate dehydrogenase represents one of the key innovations in the field of rapid diagnostics for malaria. This study explored the generation of aptamers that specifically target P. falciparum. Two sets of aptamers with diagnostically-supportive functions were generated independently, through parallel SELEX of recombinantly-expressed, full-length Plasmodium falciparum lactate dehydrogenase (rPfLDH), and an oligopeptide comprising the P. falciparum-specific epitope on lactate dehydrogenase (LDHp). The latter offers a promising solution for generating aptamers capable of binding with high specificity to P. falciparum. In this work, an rLDH class of aptamers was generated when SELEX was performed using the full-length rPfLDH protein as the target and the LDHp class of aptamers was generated when SELEX was performed using the oligopeptide LDHp as a target. Aptamers were successfully generated through the process of SELEX (systematic evolution of ligands through exponential enrichment) following the study and application of several optimisation steps, particularly during the amplification stage of SELEX. Optimisation steps included the study of improvements in PCR conditions; role of surfactants (Triton-X), modifying the PCR clean-up protocol; and agarose gel excision. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers both reported here and previously published, confirming their importance in recognition of the target. Novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Clades of consensus sequences were identified in both the rLDH and LDHp groups of aptamers, where sequences in the rLDH clade did not show preferential binding to rPfLDH while those in the LDHp clade (particularly LDHp 3 and 18) were able to recognise and bind only LDHp. Of the 19 sequences returned from the parallel SELEX procedures for rPfLDH (11 sequences) and LDHp (8 sequences), six rPfLDH and all eight LDHp sequences underwent preliminary screening and those with low responses eliminated. Of the eight LDHp-targeting aptamer sequences, five were preliminarily shown to bind to LDHp, whereas only two rPfLDH-targeting sequences were shown to bind to the target (rLDH 4 and 7). To this small selection of rPfLDH oligonucleotide sequences, two more (rLDH 1 and 15) were chosen for further study based on their sequences, secondary and predicted tertiary conformations. Sequences chosen for further study were therefore: rLDH 1, 4, 7 and 15 in the rLDH class, and LDHp 1, 3, 11, 14 and 18 in the LDHp class. Binding properties of the aptamers towards their targets were investigated using enzyme-linked oligonucleotide assays (ELONA), fluorophore-linked oligonucleotide assays (FLONA), electromobility shift assays (EMSA), surface plasmon resonance (SPR), and GelRed dissociation assays, while applications towards aptasensors were explored using electrochemical impedance spectroscopy (EIS) and fluorescent microscopy. Some inconsistencies were seen for specific aptamer to target binding interactions using specific techniques; however, generally, binding to the targets was observed across the techniques assessed. These varied responses demonstrate the need to screen and validate aptamers using a variety of techniques and platforms not necessarily specific for the proposed application. From the aptamer binding screening studies using ELONA, the most promising aptamers generated were identified as LDHp 11, rLDH 4, rLDH 7 and rLDH 15. Aptamer rLDH 4, which was generated against rPfLDH, exhibited preferential and specific binding to the lactate dehydrogenase from P. falciparum, over the recombinantly-expressed lactate dehydrogenase from Plasmodium vivax (rPvLDH), albeit with lowered responses compared to LDHp 11 in ELONA and EMSA studies. However, in kinetic ELONA studies rLDH 4 showed binding to both rPfLDH and rPvLDH. Aptamer rLDH 7 showed high affinity for rPfLDH and rPvLDH in kinetic studies using ELONA. However, screening studies with ELONA indicates that aptamer rLDH 7 may not be suitable for diagnostic tests in serum samples given its non-specific binding to human serum albumin (HSA). Aptamer rLDH 15 exhibited species specificity for rPfLDH in screening studies using ELONA but showed affinity towards rPvLDH (albeit lower relative to its affinity for rPfLDH) in kinetic studies using ELONA. LDHp 11, generated against the PfLDH peptide, showed a clear preference for rPfLDH when compared to rPvLDH and other control proteins, in both sets of ELONA studies conducted, as well as EMSA, thus possessing a strong ability to identify the presence of Plasmodium falciparum owing to its generation against the species-specific epitope. While LDHp 1 demonstrated binding to plasmodial LDH in a flow-through system (SPR), so reiterating ELONA responses, it did not perform well in the remaining methodologies. Aptamers rLDH 1 and 15 and LDHp 3, 14 and 18 exhibited a mixed set of results throughout the target protein screening analyses and were, thus, not considered for selective binding in P. falciparum parasite bodies. In studies aimed at exploring biosensor assemblies utilising the developed aptamers, both rLDH 4 and LDHp 11, along with rLDH 7, LDHp 1 and pL1, demonstrated in situ binding to the native PfLDH in fluorescent microscopy. LDHp 11 exhibited FITC-based fluorescence equivalent to the anti-rPfLDHp IgY antibody in confocal fluorescent microscopy indicating superior binding to the native PfLDH compared to the remaining aptamers. An examination of electrochemical impedance as a platform for a biosensor assembly did not, in these studies, exhibit the required sensitivity using physiologically relevant concentrations of analyte expected for pLDH following infection with Plasmodium spp. Malstat/LDH activity was explored for application in a colorimetric aptasensor. A decrease in both rPfLDH and rPvLDH activity was observed following incubation with the tested aptamers, but rLDH 1, rLDH 7 and LDHp 14 did not exhibit similar decreases in rPvLDH activity. Aptamers rLDH 1, 4 and 7 and LDHp 11 and 14 were, therefore, not selected as candidates for LDH capture in LDH activity-based diagnostic devices for P. falciparum. The decreases in pLDH activity in the presence of aptamers could hold promise as direct or antagonistic malaria therapeutic agents. Preliminary studies on the application of aptamers as malaria therapeutic agents, while of interest, should be viewed with due caution given the challenges of aptamers reaching the intracellular native plasmodial LDH hosted within the red blood cells. In conclusion, this work has shown the ability of the LDHp 11 aptamer, generated in these studies, to selectively bind rPfLDH over rPvLDH, and to bind to the native PfLDH in fluorescent microscopy, indicating that this aptamer holds promise as a biorecognition element in malaria biosensors and other diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax. The use of a species-specific epitope of P. falciparum as a target in aptamer generation paves the way for similar such studies aimed at generating aptamers with species selectivity for other Plasmodium species.
- Full Text:
- Date Issued: 2020
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