The exported protein PbCP1 localises to cleft-like structures in the rodent malaria parasite Plasmodium berghei

Protein export into the host red blood cell is one of the key processes in the pathobiology of the malaria parasite Plasmodiumtrl falciparum, which extensively remodels the red blood cell to ensure its virulence and survival. In this study, we aimed to shed further light on the protein export mechanisms in the rodent malaria parasite P. berghei and provide further proof of the conserved nature of host cell remodeling in Plasmodium spp. Based on the presence of an export motif (R/KxLxE/Q/D) termed PEXEL (Plasmodium export element), we have generated transgenic P. berghei parasite lines expressing GFP chimera of putatively exported proteins and analysed one of the newly identified exported proteins in detail. This essential protein, termed PbCP1 (P. berghei Cleft-like Protein 1), harbours an atypical PEXEL motif (RxLxY) and is further characterised by two predicted transmembrane domains (2TMD) in the C-terminal end of the protein. We have functionally validated the unusual PEXEL motif in PbCP1 and analysed the role of the 2TMD region, which is required to recruit PbCP1 to discrete membranous structures in the red blood cell cytosol that have a convoluted, vesico-tubular morphology by electron microscopy. Importantly, this study reveals that rodent malaria species also induce modifications to their host red blood cell

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions

Plasmodium falciparum: Analysis of Protein-Protein Interactions of the 140/130/110-kDa Rhoptry Protein Complex Using Antibody and Mouse Erythrocyte Binding Assays

The high-molecular-weight rhoptry proteins of Plasmodium falciparum exist in a multiprotein complex consisting of proteins of 140, 130, and 110 kDa. The complex of rhoptry proteins binds to human and mouse erythrocyte membranes in association with a 120-kDa SERA protein. These proteins are believed to participate in the process of erythrocyte invasion. We have used six different antibodies (polyclonal and monoclonal) known to precipitate the high-molecular-weight rhoptry protein complex (HMWC) to analyze the structural relationship of proteins within the complex. Limited proteolysis of immune complexes (IC) immobilized on Sepharose beads (protein \u27footprinting\u27) and binding of SV8 protease generated peptides to intact mouse erythrocytes was performed. The 140-kDa polypeptide was more susceptible to protease digestion followed by the 130- and 110-kDa polypeptides. The susceptibility of the 140-kDa polypeptide to protease digestion was independent of the type of precipitating antibody. We identified a 120-kDa protein as the major proteolytic fragment of the 140-kDa protein. SV8 protease generated peptide fragments derived from the 110- and 130-kDa proteins contained putative mouse erythrocyte binding domains. Immunoprecipitation of SV8-generated peptides gave peptide profiles similar to those obtained with protein \u27footprinting\u27. Additional experiments performed to investigate the stability of the HMWC using chaotropic and lyotropic agents demonstrated that the HMWC was stable to perturbatory reagents known to disaggregate macromolecular complexes. Solubilization of schizonts with 6 M urea and 4 M MgCl followed by IC formation led to differential precipitation of the 110-kDa polypeptide, while solubilization with 3 M KCl resulted in the differential precipitation of the 140- and 130-kDa polypeptides, suggesting that both proteins may be in direct association. Treatment of immobilized IC with different perturbatory agents including 6 M urea, 3 M KCl, 4 M MgCl , or 2% SDS from an insoluble matrix resulted in the elution of the intact complex. The mouse erythrocyte binding property of the HMWC is conserved among different geographical isolates of P. falciparum. The results provide insights concerning the mechanism of protein-protein interaction within the complex. © 1993 Academic Press, Inc. 2

The Role of Host Inflammatory Mediators As Biomarkers for Malaria Pathogenesis

Malaria pathogenesis results from a complex interplay of pro-inflammatory and antiinflammatory mediators produced as a result of innate responses following Plasmodium infection. These mediators interact with host tissues and organs resulting in damage and destruction that characterizes specific syndromes associated with red blood cell destruction, vascular endothelial disturbances, microvascular obstruction, respiratory distress, retinopathy, inflammatory responses to toxins and other parasite metabolites and placental tissue destruction. The malaria syndromes define uncomplicated malaria (UM), cerebral malaria (CM), placental malaria (PM), severe malarial anemia (SA), and severe malaria (SM). Approximately 515 million cases of malaria are reported annually with 1-3 million deaths occurring in children. Five Plasmodium species infect humans: P. falciparum, P. vivax P. malariae P. ovale and P. knowlesi. Plasmodium falciparum is the agent most associated with severe and fatal malaria. Pregnant women and children make up the most vulnerable groups to develop severe pathogenesis from malaria infection. Cytokines, chemokines, vascular endothelial proteins, angiogenic factors, and other host mediators make up important mediators of pathogenesis in the host. In addition, other non-immunologic host molecules, parasite proteins, metabolites and toxins may contribute to severe pathogenesis in the host. Determining which of the mediators enhance adverse outcomes for disease progression and poor disease prognosis as well as identifying those mediators with protective effects is an area of intense investigation in malaria research. The cytokines tumor necrosis factor alpha (TNFα), interleukin (IL)-12, IL-1ß, IL-10 and interferon gamma (IFNγ) are associated with the pathogenesis of cerebral malaria and severe anemia, with TNFα being strongly implicated with this form of malaria. Treatment of patients with neutralizing anti-TNFα antibodies was effective in resolving clinical symptoms. Low ratios of IL-10 to TNFα in serum correlate with severe anemia in contrast to high IL-10 to TNFα ratios seen in children with uncomplicated malaria. Levels of soluble ICAM-1, IL-1Ra, IL-2R, IL-4, IL-6, IL-6R, neopterin, angiopoeitin (ANG) 2 and TNFα are found to be highly elevated in patients with cerebral malaria, with TNFα being highly elevated in patients who died from cerebral malaria compared to patients with uncomplicated malaria. TNFα and ANG2 levels can predict poor prognosis with fatal outcomes in cases of cerebral malaria. A diagnostic test that is prognostic and capable of discriminating among the different malaria syndromes as well as distinguishing infections caused by non malaria pathogens will improve disease management of malaria and disease surveillance. A diagnostic test that incorporates host mediators of disease as well as parasite products and toxins would detect biomarkers of disease progression, diseases severity and development of cerebral malaria and will also be useful in determining the effectiveness of parasite clearance following drug treatment. In this chapter we examine studies performed to evaluate host mediators associated with malaria pathogenesis; uncomplicated malaria (UM), cerebral malaria (CM), placental malaria (PM), severe malarial anemia (SA), and severe malaria (SM) to determine the effectiveness of the mediators as biomarkers for malaria diagnosis. © 2012 Nova Science Publishers, Inc. All rights reserved