Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.

Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

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.

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Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted