Hosam Shams-Eldin

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase.

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.

Genes for Glycosylphosphatidylinositol Toxin Biosynthesis in Plasmodium falciparum

ABSTRACT About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.

Identification of the Archaeal alg7 Gene Homolog (Encoding N-Acetylglucosamine-1-Phosphate Transferase) of the N-Linked Glycosylation System by Cross-Domain Complementation in Saccharomyces cerevisiae

The Mv1751 gene product is thought to catalyze the first step in the N-glycosylation pathway in Methanococcus voltae. Here, we show that a conditional lethal mutation in the alg7 gene (N-acetylglucosamine-1-phosphate transferase) in Saccharomyces cerevisiae was successfully complemented with Mv1751, highlighting a rare case of cross-domain complementation.

The Trypanosoma brucei sphingolipid synthase, an essential enzyme and drug target ☆

Sphingolipids are important components of eukaryotic membranes, particularly the plasma membrane, and are involved in a diverse array of signal transduction processes. In the Eukaryota the biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals which produce sphingomyelin (SM), several pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. This process is catalyzed by the enzyme IPC synthase, a recognized target for anti-fungals encoded by the AUR1 gene in yeast. Recently, functional orthologues of the AUR1p have been identified in a group of insect vector-borne pathogenic protozoa, the Kinetoplastida, which are responsible for a range of so-called neglected diseases. Of these the Trypanosoma brucei species are the causative agents of human African trypanosomiasis in many of the most under-developed regions of Africa. The available treatments for these diseases are limited, of decreasing efficacy, and often demonstrate severe side-effects. Against this background the T. brucei sphingolipid synthase, an orthologue of the yeast AUR1p, may represent a promising target for novel anti-protozoals. Our studies identify an isoform of this protein as a novel bi-functional enzyme capable of catalyzing the synthesis of both IPC and SM, both known to be present in the parasite. Furthermore, the synthase is essential for parasite growth and can be inhibited by a known anti-fungal at low nanomolar levels in vitro. Most notably this drug demonstrates trypanocidal activity against cultured bloodstream form parasites. Thus, the T. brucei sphingolipid synthase represents a valid and promising drug target.

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.

Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus‐like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non‐phosphorylatable form of VP40 (VP40mut). VP40mut was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon‐induced gene expression similarly to wild‐type VP40. However, VP40mut was specifically impaired in its ability to recruit nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of nucleocapsids to the viral envelope.

The Dual Origin of Toxoplasma gondii N-Glycans

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins.Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.

Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii

Graphical abstract Highlights ► Identification and characterisation of Toxoplasma sphingolipid synthase (TgSLS). ► Demonstration of TgSLS inositol phosphorylceramide synthase activity. ► Identification of inositol phosphorylceramide in Toxoplasma extracts. ► Delineation of role of host sphingolipid biosynthesis in Toxoplasma proliferation. ► Host biosynthesis non-essential for proliferation, de novo synthesis could be key.