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The Pathogenic Diplomanad Giardia and the Parabasalid Trichomonas

The diplomonad Giardia is an exceedingly common waterborne intestinal parasite responsible for global disease, with a chronic burden particularly in children in developing countries [97, 98]. The parasite has the distinctive cellular signatures of a flattened pear shape, concave ventral disk, and two anterior nuclei in the tro-phozoites stage (four nuclei in the transmissible nonmotile cyst stage). It has a relatively simplified cellular organization but retains an endoplasmic reticulum, Golgi apparatus, and a "rudimentary" organelle termed a mitosome (described further below). Giardia possesses both anterior and posterior flagella, as well as two recurrent dorsal flagella. The parasite adheres to intestinal epithelial cells using the unique concave ventral disk that creates a mechanical suction via contraction of myosin-actin filaments, perhaps aided by the beating of the recurrent dorsal flagella. Feeding occurs via pinocytosis from the intestinal lumen at the dorsal face of the parasite.

Giardia and the apicomplexan Cryptosporidium share two notable characteristics: a highly evolved scavenging parasitism resulting in the loss of multiple metabolic pathways; and an "anaerobic" lifestyle, indicated by the absence of many mitochondrial functions, including lack of the Embden-Meyerhoff pathway and mitochondrial electron transport (reviewed in Ref. [97], see also Ref. [4]). Both parasites have an external environmentally durable cyst stage, but whereas Cryp-tosporidium invades host intestinal epithelial cells and resides within a parasito-phorous vacuole, Giardia remains extracellular throughout its life cycle. This difference might be reflected in the respective catalogs of lineage-specific surface proteins: no adhesive surface proteins have been identified in Giardia, and the large repertoire of cysteine-rich variant surface antigen proteins (vsps) probably function solely in protection from the harsh intestinal environment and evasion of mucosal immune surveillance [99, 100]. The complete nucleotide sequence for Giardia lamblia has been submitted to GenBank (Table 19.1; see www.mbl.edu/ Giardia), but no final project summary has been published at the time of this writing.

Parabasalids, such as Trichomonas vaginalis, are also typically "anaerobic," and some members are pathogenic in the urogenital or intestinal tract. They possess three or more basal flagella plus a recurrent membrane-associated flagellum, most notably in Trichomonas, that creates an undulating wave. Parabasalids are also noteworthy for the presence of acristate dense granules termed hydrogeno-somes that are involved in pyruvate metabolism leading to the generation of molecular hydrogen [101-103]. The parasite lacks a cyst stage and is dependent on sexual transmission [104]. A Trichomonas genome sequencing project is nearing completion (Table 19.1) [105], and it is hoped that proteome annotation will reveal insights into the hydrogenosome structure, metabolic pathways, and tissue-invasive and tissue-adhesive mechanisms of this pathogenic protozoan. Comparative genomics might reveal common themes in the adaptations to anaerobic, parasitic lifestyles in Trichomonas, Giardia, and Cryptosporidium. For example, the Crypto-sporidium "remnant" mitochondrion and the Giardia "mitosome" both appear to be sites of iron-sulfur protein maturation [4, 106, 107]. Little is known regarding Trichomonas virulence factors, and to date the most prominent studies center on a possibly large family of cysteine proteinases [108-110], adhesins [111], and a possibly large family of surface proteins containing a leucine-rich repeat [112], all predicted to be involved in tissue invasion and epithelial adhesion.

Giardia and Trichomonas have been traditionally regarded as "early branching" eukaryotes, based upon the widespread and apparently erroneous belief that they lack a mitochondrion, supporting the attractive hypothesis that this indicates an evolutionary branching prior to the appearance of this organelle in eukaryotes. Although their early branching status may yet hold true, as suggested from phyloge-netic analyses of select genes [113-115], it has recently been realized that the vestigial or absent mitochondrion is either the result of a secondary loss or remains as a highly evolved diminutive organelle, as evidenced by nuclear genes of apparent

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mitochondrial or a-proteobacterial origin [107, 116-118]. Thus it is apparent that all known extant eukaryotes share a common ancestor having both a mitochondrion and nucleus. To date phylogenies have been based upon the presence or absence of specific traits, such as morphological structures, diagnostic gene fusions, or diagnostic insertions within genes, or analyses based upon the phylo-genetic trees of discrete genes (for an excellent discussion see Ref. [19]). However, any approach that is reliant on discrete evolutionary events is prone to errors arising from convergence or lateral gene transfer; and it is therefore hoped that phylogenies based upon whole-genome analyses of sequence data from multiple protozoans, both pathogenic and free-living, will refine our understanding of the "last common ancestor" of eukaryotes as well as delineate the branching of the protozoans and place the protozoans in proper phylogenetic order with respect to the metazoans, fungi, and plants. More problematic is rooting Giardia and Trichomonas at the base of the protozoan tree, but whole-genome-based comparisons of protozoans, bacteria, and archaea might indeed confirm their place among the earliest branching protozoans. Once a protozoan phylogenetic tree is put in place, we can then address with confidence the role of endosymbiotic events and nuclear gene transfer, independent lateral gene transfer, and convergence in the evolution of the protozoans and adaptations conferring parasitism.

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