IMMUNE EVASION AND THE EVOLUTION OF MOLECULAR MIMICRY IN PARASITES

Parasites that are molecular mimics express proteins which resemble host proteins. This resemblance facilitates immune evasion because the immune molecules with the specificity to react with the parasite also cross‐react with the host's own proteins, and these lymphocytes are rare. Given this advantage, why are not most parasites molecular mimics? Here we explore potential factors that can select against molecular mimicry in parasites and thereby limit its occurrence. We consider two hypotheses: (1) molecular mimics are more likely to induce autoimmunity in their hosts, and hosts with autoimmunity generate fewer new infections (the “costly autoimmunity hypothesis”); and (2) molecular mimicry compromises protein functioning, lowering the within‐host replication rate and leading to fewer new infections (the “mimicry trade‐off hypothesis”). Our analysis shows that although both hypotheses may select against molecular mimicry in parasites, unique hallmarks of protein expression identify whether selection is due to the costly autoimmunity hypothesis or the mimicry trade‐off hypothesis. We show that understanding the relevant selective forces is necessary to predict how different medical interventions will affect the proportion of hosts that experience the different infection types, and that if parasite evolution is ignored, interventions aimed at reducing infection‐induced autoimmunity may ultimately fail.     

Erythrocyte invasion: vocabulary and grammar of the Plasmodium rhoptry

Malaria is a dangerous infectious disease caused by obligate intracellular protozoan Plasmodium parasites. In the vertebrate host, erythrocyte recognition and establishment of a nascent parasitophorous vacuole are essential processes, and are largely achieved using molecules located in the microorganelles of the invasive-stage parasites. Recent proteome analyses of the phylogenetically related Toxoplasma parasite have provided protein catalogs for these microorganelles, which can now be used to identify orthologous proteins in the Plasmodium genome. Of importance is the formation of a complex between the proteins secreted from the rhoptry neck portion (RONs) and micronemes (AMA1), which localize at the moving junction during parasite invagination into the host cell. In this article I review the largely unexplored paradigm of the malaria merozoite rhoptry, focusing on the high molecular weight rhoptry protein complex (the RhopH complex), and speculate on its grammar during invasion.     

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.     

Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed “clean linear B cell epitopes,” and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.     

Schistosoma mansoni: biochemical characteristics of the antischistosomal effects of Ro 15-5458

We evaluated a variety of biochemical parameters in Schistosoma mansoni isolated from mice up to 4 days after dosing with 15 mg/kg Ro 15-5458. While no drug effect could be demonstrated in the utilization of media glucose, glycogen content, gut pigment, or ATP levels of the parasites, a significant reduction (P less than or equal to 0.05) in parasite weight and protein content was observed. Possible drug actions that may contribute to the loss in parasite protein and perhaps ultimately result in parasite death have been investigated. We noted significant reduction in the incorporation of leucine and thymidine into acid-insoluble fractions of the parasites. The reduction in the incorporation of leucine into parasite proteins was nonspecific and preceded the effect of the drug on the uptake of the amino acid. Parasite and host liver RNA isolated after dosing were translated in vitro in a rabbit reticulocyte system. Drug-treated parasite mRNA, but not that of the host, was less effective than control mRNA in directing the incorporation of [35S]methionine. We propose a hypothesis that attributes the loss in protein content to a defect in the biosynthesis of parasite proteins as a result of a drug-induced reduction in the quantity of mRNA in the parasites This effect of Ro 15-5458 on the parasite may provide the basis for its schistosomicidal action.     

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.     

Comparative proteomic analysis of Fasciola hepatica juveniles and Schistosoma bovis schistosomula

Protein interactions between host and parasites can influence the infection success and severity. The aim of this investigation was to identify the proteins from two trematodes potentially localized at the host-parasite interface. We performed the proteomic profiles from in vivo obtained immature lung stage Schistosoma bovis schistosomula and in vitro excysted juveniles from Fasciola hepatica, parasites of ruminants and man usually giving rise to chronic infections. Proteomes from those parasites were obtained after digestion with trypsin and the peptides generated were identified by mass spectrometry, both before and after parasites' treatment with 70% methanol. The comparison of the two proteome sets from each parasite and between them, the analysis of their relative abundance and of their potential exposure to the host from living parasites, together with the specific immunolocalization of two of the identified molecules, show that this approach could assist in the identification of parasite exposed proteins and in the definition of molecules common for the two parasites with potential interaction with the host. Further characterization of these molecules could guide to define new common anti-parasitic targets and potential vaccine candidates.     

Labeling strategies to track protozoan parasite proteome dynamics

Intracellular protozoan parasites are responsible for wide-spread infectious diseases. These unicellular pathogens have complex, multi-host life cycles, which present challenges for investigating their basic biology and for discovering vulnerabilities that could be exploited for disease control. Throughout development, parasite proteomes are dynamic and support stage-specific functions, but detection of these proteins is often technically challenging and complicated by the abundance of host proteins. Thus, to elucidate key parasite processes and host–pathogen interactions, labeling strategies are required to track pathogen proteins during infection. Herein, we discuss the application of bioorthogonal non-canonical amino acid tagging and proximity-dependent labeling to broadly study protozoan parasites and include outlooks for future applications to study Plasmodium, the causative agent of malaria. We highlight the potential of these technologies to provide spatiotemporal labeling with selective parasite protein enrichment, which could enable previously unattainable insight into the biology of elusive developmental stages.     

Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.     

Mapping the human proteome for non-redundant peptide islands

We describe immune-proteome structures using libraries of protein fragments that define a structural immunological alphabet. We propose and validate such an alphabet as i) composed of letters of five consecutive amino acids, pentapeptide units being sufficient minimal antigenic determinants in a protein, and ii) characterized by low-similarity to human proteins, so representing structures unknown to the host and potentially able to evoke an immune response. In this context, we have thoroughly sifted through the entire human proteome searching for non-redundant protein motifs. Here, for the first time, a complete sequence redundancy dissection of the human proteome has been conducted. The non-redundant peptide islands in the human proteome have been quantified and catalogued according to the amino acid length. The library of uniquely occurring n-peptide sequences that was obtained is characterized by a logarithmic decrease of the number of non-redundant peptides as a function of the peptide length. This library represents a highly specific catalogue of molecular protein signatures, the possible use of which in cancer/autoimmunity research is discussed, with a major focus on non-redundant dodecamer sequences.     

Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.     

Searching for local similarities between HIV-1 and human proteins. Application to vaccines

A method for identification of fragments with a high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions, whose fragments are frequent in human proteins, and regions, exhibiting a high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV-infection due either to induction of cross-reacting effectors of immune system or through molecular mimicry of physiologically important human proteins, leading to an alteration of homeostasis of the organism, is discussed. Most of regions, identified in HIV-1 proteins, contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines which enable the exclusion of epitopes, having the (immuno)pathogenic potential, are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions, whose fragments are frequent in human proteins, as well as of regions exhibiting a pronounced local similarity to proteins that provide for important physiological functions.     

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.     

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.     

Searching for Local Similarities between HIV-1 and Human Proteins. Application to Vaccines

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8⁺ CTL or CD4⁺ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.     

Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.     

Expression variance,biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7

During intracellular stay, Toxoplasma gondii secretes dense granule proteins (GRA) which remodel the parasitophorous vacuole and are considered functional in parasite-host interrelation. Comparative analysis of parasites from mouse-virulent strain BK and an in vitro attenuated variant revealed that the level of GRA7 expression correlates with T. gondii virulence: proteome analysis and quantitation by immunoblot demonstrated a massive decrease in GRA7 steady-state synthesis parallel to the loss of virulence. Properties of GRA7 that are pertinent to its membrane targeting and to GRA7-directed immune resistance were studied in detail. GRA7 is exclusively membrane-associated in both parasites and infected host cells as demonstrated by subcellular fractionations. Triton X-114 partitioning of isolated parasites substantiated that GRA7 is an integral membrane protein, the hydrophobic stretch from amino acid 181 to 202 providing a possible membrane anchor. A fraction enriched for membranous material from infected host cells contained additional forms of GRA7 with reduced mobility in gel electrophoresis, indicating that the protein is modified after exocytosis from the parasite. By flow cytometric analysis, GRA7 was detected on the surface of intact host cells. An intracellular origin of surface-associated GRA7 seems likely since GRA7 released from extracellular parasites failed to label the host cell surface. Consistent with a role at a parasite-host interface, GRA7 proved to be a target antigen of the intracerebral immune response as evidenced by the presence of GRA7-specific antibodies in mouse cerebrospinal fluid during chronic infection.     

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.     

How viruses hijack cell regulation

Viruses, as obligate intracellular parasites, are the pathogens that have the most intimate relationship with their host, and as such, their genomes have been shaped directly by interactions with the host proteome. Every step of the viral life cycle, from entry to budding, is orchestrated through interactions with cellular proteins. Accordingly, viruses will hijack and manipulate these proteins utilising any achievable mechanism. Yet, the extensive interactions of viral proteomes has yielded a conundrum: how do viruses commandeer so many diverse pathways and processes, given the obvious spatial constraints imposed by their compact genomes? One important approach is slowly being revealed, the extensive mimicry of host protein short linear motifs (SLiMs).