Human serum haptoglobin is toxic to Plasmodium falciparum in vitro

Innate immune responses are important in the control of malaria, particularly in those who have not yet mounted an effective adaptive response. Here we report that the human serum acute phase protein, haptoglobin is toxic to Plasmodium falciparum cultured in vitro. This effect is phenotype-dependent and occurs during the trophozoite phase of the asexual life cycle. We propose that the increased levels of haptoglobin seen in the acute phase response may be protective against malaria in humans

Plasmodium falciparum exported protein PFE60 influences Maurer's clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer's clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore - unlike all other described resident MC membrane proteins - PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.This work was supported by the Australian Research Council (DP1093518 and DP0878953)

Antigenic variation in Plasmodium falciparum : understanding the RIFIN protein family

RIFIN proteins are variable surface antigens, which have a central role in the survival and virulence of the malaria parasite Plasmodium falciparum. Antigenic variation is a mean for these parasites to avoid clearance by the host s immune system. However, this is often a secondary function to the main role of these proteins. In the case of RIFIN, P. falciparum s largest multicopy protein family, the main functions remain unknown. In order to elucidate a protein s function, it is crucial to understand its basic properties, including the structure of the protein family, their localization and the protein s topology. Through different methods, we have strived to simplify the RIFIN protein family into manageable entities. We have started with a simple classification of RIFIN proteins into meaningful sub-groups. We have predicted that these sub-groups are functionally distinct, although they probably share a related function. We then designed RSPred, an automatic method, based on hidden Markov models and a sorting program, to detect and classify RIFIN and STEVOR sequences according to their sub-group. Finally, using an in vitro method to determine protein topology, we have analyzed both A-RIFIN and B-RIFIN proteins for their number of transmembrane segments and their topologies. We show that both protein groups have a signal sequence targeting them to lipid bilayers and only one transmembrane domain. They both share a common topology where the bulk of the protein is exposed to the extracellular environment. With the current knowledge of RIFIN protein localizations, as well as the loss of expression of A-RIFIN but not B-RIFIN proteins in a splenectomized patient, it seems increasingly clear that B-RIFIN proteins are good targets for future studies, to decipher the functions of these variable proteins

Characterization of novel malaria vaccine candidates representing alpha-helical coiled coil domains

The future vision in the battle against malaria goes beyond controlling the disease. Envisioned is the world-wide eradication of malaria. A substantial contribution to reach this goal is the development of an effective vaccine. Today’s most advanced and most effective malaria vaccine, RTS,S/AS01, showed efficacy of 30 to 66% against all clinical episodes. There is a great need to increase efficacy by the next generation malaria vaccines. A strategy for increasing RTS,S efficacy could be to combine it with an effective blood stage vaccine. The disappointing outcomes of clinical trials conducted for most current blood stage vaccines demands the identification of novel promising candidates. Under persistent exposure individuals develop immunity that protects against clinical disease but not parasitemia. This natural acquired immunity develops slowly and is reached in adolescence. In contrast, immunity against severe disease develops already after few infections. The mechanisms that underlie naturally acquired immunity or severe disease immunity remain poorly understood. Antibodies were demonstrated to play a critical role for controlling blood stage infection. It remains unclear which proteins elicit the production of protective antibodies and through which antibody effector function protection is provided. The relevance of antibodies in blood stage protection has the consequence that the immunogen correctly mimic the three-dimensional structure of the native protein. This PhD thesis has its major focus on immunogens that adopt a stable tertiary structure in aqueous environment. The availability of the P. falciparum genome sequences, transcriptome and proteome data has opened the avenue for the identification of novel targets for vaccine development. However, blood stage vaccine development has focused on only a few candidates. Previously our collaborators in this project have identified promising candidates by genome-wide screening for alpha-helical coiled coil domains in proteins expressed in the erythrocytic parasite stages. The segments with high probability score for coiled coil formation where selected. The 166 coiled coil segments derived from 131 proteins representing 4% of the blood stage proteome. 95 coiled coil fragments of a length of 30-40 amino acids were synthesized and analyzed systematically in a pre-clinical evaluation pathway. The aim of this thesis was to fill the gaps in the preclinical evaluation pathway of novel synthetic peptide vaccine candidates. The extensive polymorphism found in most parasite antigens represents a major obstacle for the development of efficacious blood stage vaccines. The genetic diversity of the identified coiled coil protein segments was studied in great detail. We found that coiled coil segments are well conserved, 82% of all selected 166 segments showed complete sequence conservation. Polymorphism was found predominantly in segments containing almost perfect tandem repeats. Based on these findings an optimized bioinformatic selection strategy was formulated proposing to exclude coiled coil segments consisting of almost perfect tandem repeats. The availability of basic knowledge about vaccine candidates is a prerequisite for vaccine development and is essential to attract further funding for continued clinical development. A detailed cell biological characterization was undertaken for the most promising candidate, PFF0165c (newly termed Trophozoite exported protein 1 (Tex1)) Transcript and protein levels were analyzed throughout the intra-erytrocytic development cycle. Tex1 transcripts were found up-regulated in the early trophozoite stage. This was supported by Tex1 protein levels. Tex1 abundance persisted until parasite egress. Immunofluorescence experiments revealed that Tex1 is exported and associates to parasite-derived structures, termed Maurer’s clefts. Before parasite egress Tex1 resides in close proximity to the red blood cell membrane. In the search of sequence motifs responsible for Tex1 export we found that the actual translational start site is positioned 43 amino acids upstream of the start site previously predicted. The additional 43 amino acids function as signal peptide, directing the protein into the classical secretory pathway. This thesis contributed to the immunological characterization of the intrinsically unstructured region (P27A) of Tex1. P27A was evaluated for vaccine potential and met the principal requirements to be downselected for a phase 1 trial. P27A was recognized by a majority of naturally exposed individuals, highly immunogenic, highly conserved and P27A-specific human and mouse sera were effective in in vitro parasite killing by Antibody-dependent cellular inhibition assay. High P27A-specifc antibody titers were found to positively correlate with protection. Clinical grade P27A peptide is currently produced. In order to validate synthetic peptides as antigens the recognition by sera of adults from endemic region was compared to the recognition of the antigen recombinant expressed in E. coli. Comparable recognition of both types of antigens was observed. This thesis provides evidence that the approach initiated by our collaborators is invaluable. This strategy, if proven successful in clinical trials, could be applied for vaccine development against many other pathogens from which genome data is available

Evaluation of a videotape designed to reduce computer anxiety in preservice teachers

http://www.worldcat.org/oclc/1132106

Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer\u27s clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process

Cellular automata for dynamic S-boxes in cryptography.

In today\u27s world of private information and mass communication, there is an ever increasing need for new methods of maintaining and protecting privacy and integrity of information. This thesis attempts to combine the chaotic world of cellular automata and the paranoid world of cryptography to enhance the S-box of many Substitution Permutation Network (SPN) ciphers, specifically Rijndael/AES. The success of this enhancement is measured in terms of security and performance. The results show that it is possible to use Cellular Automata (CA) to enhance the security of an 8-bit S-box by further randomizing the structure. This secure use of CA to scramble the S-box, removes the 9-term algebraic expression [20] [21] that typical Galois generated S-boxes share. This cryptosystem securely uses a Margolis class, partitioned block, uniform gas, cellular automata to create unique S-boxes for each block of data to be processed. The system improves the base Rijndael algorithm in the following ways. First, it utilizes a new S-box for each block of data. This effectively limits the amount of data that can be gathered for statistical analysis to the blocksize being used. Secondly, the S-boxes are not stored in the compiled binary, which protects against an S-box Blanking [22] attack. Thirdly, the algebraic expression hidden within each galois generated S-box is destroyed after one CA generation, which also modifies key expansion results. Finally, the thesis succeeds in combining Cellular Automata and Cryptography securely, though it is not the most efficient solution to dynamic S-boxes

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>STEVOR proteins, encoded by the multicopy <it>stevor </it>gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.</p> <p>Methods</p> <p>Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.</p> <p>Results</p> <p>Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.</p> <p>Conclusion</p> <p>The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.</p