Christian Pick

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.

The occurrence of hemocyanin in Hexapoda.

Hemocyanins are copper‐containing, respiratory proteins that have been thoroughly studied in various arthropod subphyla. Specific O2‐transport proteins have long been considered unnecessary in Hexapoda (including Insecta), which acquire O2 via an elaborate tracheal system. However, we recently identified a functional hemocyanin in the stonefly Perla marginata (Plecoptera) and in the firebrat Thermobia domestica (Zygentoma). We used RT‐PCR and RACE experiments to study the presence of hemocyanin in a broad range of ametabolous and hemimetabolous hexapod taxa. We obtained a total of 12 full‐length and 5 partial cDNA sequences of hemocyanins from representatives of Collembola, Archeognatha, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera and Blattaria. No hemocyanin could be identified in Protura, Diplura, Ephemeroptera, Odonata, or in the Eumetabola (Holometabola + Hemiptera). It is not currently known why hemocyanin has been lost in some taxa. Hexapod hemocyanins usually consist of two distinct subunit types. Whereas type 1 subunits may represent the central building block, type 2 subunits may be absent in some species. Phylogenetic analyses support the Pancrustacea hypothesis and show that type 1 and type 2 subunits diverged before the emergence of the Hexapoda. The copperless insect storage hexamerins evolved from hemocyanin type 1 subunits, with Machilis germanica (Archeognatha) hemocyanin being a possible ‘intermediate’. The evolution of hemocyanin subunits follows the widely accepted phylogeny of the Hexapoda and provides strong evidence for the monophyly of the Polyneoptera (Plecoptera, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera, Blattaria) and the Dictyoptera (Mantodea, Isoptera, Blattaria). The Blattaria are paraphyletic with respect to the termites.

The diversity and evolution of chelicerate hemocyanins

BackgroundOxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as hemocyanins. Arthropod hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O2 transport capacity and a high cooperativity, thereby enhancing O2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of hemocyanins and the emergence of the large hemocyanin oligomers, hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes.ResultsHemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders.ConclusionFormation of a complex hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of hemocyanins for such approach.

Phylogenomic analyses of malaria parasites and evolution of their exported proteins

BackgroundPlasmodium falciparum is the most malignant agent of human malaria. It belongs to the taxon Laverania, which includes other ape-infecting Plasmodium species. The origin of the Laverania is still debated. P. falciparum exports pathogenicity-related proteins into the host cell using the Plasmodium export element (PEXEL). Predictions based on the presence of a PEXEL motif suggest that more than 300 proteins are exported by P. falciparum, while there are many fewer exported proteins in non-Laverania.ResultsA whole-genome approach was applied to resolve the phylogeny of eight Plasmodium species and four outgroup taxa. By using 218 orthologous proteins we received unanimous support for a sister group position of Laverania and avian malaria parasites. This observation was corroborated by the analyses of 28 exported proteins with orthologs present in all Plasmodium species. Most interestingly, several deviations from the P. falciparum PEXEL motif were found to be present in the orthologous sequences of non-Laverania.ConclusionOur phylogenomic analyses strongly support the hypotheses that the Laverania have been founded by a single Plasmodium species switching from birds to African great apes or vice versa. The deviations from the canonical PEXEL motif in orthologs may explain the comparably low number of exported proteins that have been predicted in non-Laverania.

Phylogeny of haemosporidian blood parasites revealed by a multi-gene approach

The apicomplexan order Haemosporida is a clade of unicellular blood parasites that infect a variety of reptilian, avian and mammalian hosts. Among them are the agents of human malaria, parasites of the genus Plasmodium, which pose a major threat to human health. Illuminating the evolutionary history of Haemosporida may help us in understanding their enormous biological diversity, as well as tracing the multiple host switches and associated acquisitions of novel life-history traits. However, the deep-level phylogenetic relationships among major haemosporidian clades have remained enigmatic because the datasets employed in phylogenetic analyses were severely limited in either gene coverage or taxon sampling. Using a PCR-based approach that employs a novel set of primers, we sequenced fragments of 21 nuclear genes from seven haemosporidian parasites of the genera Leucocytozoon, Haemoproteus, Parahaemoproteus, Polychromophilus and Plasmodium. After addition of genomic data from 25 apicomplexan species, the unreduced alignment comprised 20,580 bp from 32 species. Phylogenetic analyses were performed based on nucleotide, codon and amino acid data employing Bayesian inference, maximum likelihood and maximum parsimony. All analyses resulted in highly congruent topologies. We found consistent support for a basal position of Leucocytozoon within Haemosporida. In contrast to all previous studies, we recovered a sister group relationship between the genera Polychromophilus and Plasmodium. Within Plasmodium, the sauropsid and mammal-infecting lineages were recovered as sister clades. Support for these relationships was high in nearly all trees, revealing a novel phylogeny of Haemosporida, which is robust to the choice of the outgroup and the method of tree inference.

Molecular characterization of hemocyanin and hexamerin from the firebrat Thermobia domestica (Zygentoma)

Hexapods possess a tracheal system that enables the transport of oxygen to the inner organs. Although respiratory proteins have been considered unnecessary in most Hexapoda for this reason, we recently showed the presence of a functional hemocyanin in the stonefly Perla marginata. Here we report the identification and molecular characterization of a hemocyanin from Zygentoma (Thysanura). We obtained the full length cDNA of two distinct subunit types from the firebrat Thermobia domestica, and partial sequences of the orthologs from the silverfish Lepisma saccharina. The native T. domestica hemocyanin subunits both consist of 658 amino acids, but a signal peptide for transmembrane transport is missing in subunit 2. In adult firebrats both hemocyanin subunits represent a substantial proportion of the total hemolymph proteins. Phylogenetic analyses show that the subunit types are orthologous to subunits 1 and 2 of the stonefly Perla marginata. We further identified and sequenced a hexamerin subunit from T. domestica (689 amino acids), which suggests an early emergence of this type of proteins in hexapod evolution. In contrast to most other hexamerins, it does not reveal a high content in phenylalanine and tyrosine, which may be interpreted that the accumulation of aromatic amino acids commenced later in hexamerin evolution. Molecular clock calculations using hexamerins suggest that the divergence of Zygentoma and Pterygota occurred around 387 million years ago, which is in excellent agreement with the available fossil record.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera+Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.

The Plasmodium falciparum exportome contains non-canonical PEXEL/HT proteins

The pathogenicity of Plasmodium falciparum is partly due to parasite‐induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non‐canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non‐functional. Here, we show that non‐canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non‐canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non‐canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non‐canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non‐canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non‐canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.

Occurrence of Hemocyanin in Ostracod Crustaceans

Hemocyanin is a copper-containing protein that transports O2 in the hemolymph of many arthropod species. Within the crustaceans, hemocyanin appeared to be restricted to Malacostraca but has recently been identified in Remipedia. Here, we report the occurrence of hemocyanin in ostracods, indicating that this respiratory protein is more widespread within crustaceans than previously thought. By analyses of expressed sequence tags and by RT-PCR, we obtained four full length and nine partial hemocyanin sequences from six of ten investigated ostracod species. Hemocyanin was identified in Myodocopida (Actinoseta jonesi, Cypridininae sp., Euphilomedes morini, Skogsbergia lerneri, Vargula tsujii) and Platycopida (Cytherelloidea californica) but not in Podocopida. We found no evidence for the presence of hemoglobin in any of these ostracod species. Like in other arthropods, we identified multiple hemocyanin subunits (up to six) to occur in a single ostracod species. Bayesian phylogenetic analyses showed that ostracod hemocyanin subunit diversity evolved independently from that of other crustaceans. Ostracod hemocyanin subunits were found paraphyletic, with myodocopid and platycopid subunits forming distinct clades within those of the crustaceans. This pattern suggests that ostracod hemocyanins originated from distinct subunits in the pancrustacean stemline.