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

PfRH5: A Novel Reticulocyte-Binding Family Homolog of Plasmodium falciparum that Binds to the Erythrocyte, and an Investigation of Its Receptor

Multiple interactions between parasite ligands and their receptors on the human erythrocyte are a condition of successful Plasmodium falciparum invasion. The identification and characterization of these receptors presents a major challenge in the effort to understand the mechanism of invasion and to develop the means to prevent it. We describe here a novel member of the reticulocyte-binding family homolog (RH) of P. falciparum, PfRH5, and show that it binds to a previously unrecognized receptor on the RBC. PfRH5 is expressed as a 63 kDa protein and localized at the apical end of the invasive merozoite. We have expressed a fragment of PfRH5 which contains the RBC-binding domain and exhibits the same pattern of interactions with the RBC as the parent protein. Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase. We have determined the affinity, copy number and apparent molecular mass of the receptor protein. Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathwa

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

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria