Adrian B. Hehl

Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardias requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardias genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Toxoplasma gondii Homologue of Plasmodium Apical Membrane Antigen 1 Is Involved in Invasion of Host Cells

ABSTRACT Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of ∼3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A ∼54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Protein import, replication and inheritance of a vestigial mitochondrion

Mitochondrial remnant organelles (mitosomes) that exist in a range of “amitochondrial” eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in iron-sulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically.

Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest

Mitochondrial fission is mediated by dynamin-like proteins (DLPs). Trypanosoma brucei contains a single DLP, which is the only member of the dynamin superfamily. We have previously shown that expression of the human proapoptotic Bax in T. brucei induces extensive mitochondrial fragmentation. Here we report that Baxinduced mitochondrial fission is abolished in cell lines lacking functional DLP suggesting that the protein is also required for mitochondrial division during the cell cycle. Furthermore, DLP-ablated cells are deficient for endocytosis and as a consequence accumulate enlarged flagellar pockets. Thus, besides its expected role in mitochondrial fission the trypanosomal DLP is required for endocytosis, a function thought to be restricted to classical dynamins. In agreement with its dual function, the DLP localizes to both the mitochondrion and the flagellar pocket, the site where endocytosis occurs. Unexpectedly, ablation of DLP also causes an arrest of cytokinesis. The fact that no multinucleation is observed in the arrested cells argues for a precise cell-cycle block. Furthermore, analysis of a clathrin-knockdown cell line suggests that the cytokinesis arrest is not due to the endocytosis defect. Thus, our results support a working model in which mitochondrial fission triggers a checkpoint for cytokinesis.

Secretory protein trafficking in Giardia intestinalis

Early diverged extant organisms, which may serve as convenient laboratory models to look for and study evolutionary ancient features of eukaryotic cell biology, are rare. The diplomonad Giardia intestinalis, a protozoan parasite known to cause diarrhoeal disease, has become an increasingly popular object of basic research in cell biology, not least because of a genome sequencing project nearing completion. Commensurate with its phylogenetic status, the Giardia trophozoite has a very basic secretory system and even lacks hallmark structures such as a morphologically identifiable Golgi apparatus. The cells capacity for protein sorting is nevertheless unimpeded, exemplified by its ability to cope with massive amounts of newly synthesized cyst wall proteins and glycans, which are sorted to dedicated Golgi‐like compartments termed encystation‐specific vesicles (ESVs) generated from endoplasmic reticulum (ER)‐derived transport intermediates. This soluble bulk cargo is kept strictly separate from constitutively transported variant surface proteins during export, a function that is dependent on the stage‐specific recognition of trafficking signals. Encysting Giardia therefore provide a unique system for the study of unconventional, Golgi‐independent protein trafficking mechanisms in the broader context of eukaryotic endomembrane organization and evolution.

Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia

During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport.

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin‐related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi‐like delay compartments dubbed encystation‐specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal–lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant‐negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal–lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant‐negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.

A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in Giardia lamblia

ABSTRACT The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardias proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

ABSTRACT The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a β(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors.