The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target.

BACKGROUND: Giardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs. METHODS: An extensive search of the Giardia genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed in-silico to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient Saccharomyces cerevisiae strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating Giardia trophozoites. Finally, the localization of epitope-tagged and ectopically expressed GlPDE in Giardia cells was investigated. RESULTS: Giardia encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing Giardia-specific inhibitors is feasible. Recombinant GlPDE hydrolyzes cAMP with a Km of 408 µM, and cGMP is not accepted as a substrate. A number of drugs exhibit a high degree of correlation between their potency against the recombinant enzyme and their inhibition of trophozoite proliferation in culture. Epitope-tagged GlPDE localizes as dots in a pattern reminiscent of mitosomes and to the perinuclear region in Giardia. CONCLUSIONS: Our data strongly suggest that inhibition of G. lamblia PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs.

Proteomics of secretory and endocytic organelles in Giardia lamblia.

Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694.

An ER-directed transcriptional response to unfolded protein stress in the absence of conserved sensor-transducer proteins in Giardia lamblia.

The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER-overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan-dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER-folding stress. We show that on the contrary, ER-folding stress triggers a stressor-specific, ER-directed response with upregulation of only ~ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis-acting motifs in upstream regions of responder genes capable of stressor-specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function.

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

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 ß(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.

Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.

Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1-3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.

Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.

Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of ;naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes.

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes.

Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.

MAHRP-1, a novel Plasmodium falciparum histidine-rich protein, binds ferriprotoporphyrin IX and localizes to the Maurer's clefts.

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.