Genomics of Preaxostyla Flagellates Illuminates the Path Towards the Loss of Mitochondria.

The notion that mitochondria cannot be lost was shattered with the report of an oxymonad Monocercomonoides exilis, the first eukaryote arguably without any mitochondrion. Yet, questions remain about whether this extends beyond the single species and how this transition took place. The Oxymonadida is a group of gut endobionts taxonomically housed in the Preaxostyla which also contains free-living flagellates of the genera Trimastix and Paratrimastix. The latter two taxa harbour conspicuous mitochondrion-related organelles (MROs). Here we report high-quality genome and transcriptome assemblies of two Preaxostyla representatives, the free-living Paratrimastix pyriformis and the oxymonad Blattamonas nauphoetae. We performed thorough comparisons among all available genomic and transcriptomic data of Preaxostyla to further decipher the evolutionary changes towards amitochondriality, endobiosis, and unstacked Golgi. Our results provide insights into the metabolic and endomembrane evolution, but most strikingly the data confirm the complete loss of mitochondria for all three oxymonad species investigated (M. exilis, B. nauphoetae, and Streblomastix strix), suggesting the amitochondriate status is common to a large part if not the whole group of Oxymonadida. This observation moves this unique loss to 100 MYA when oxymonad lineage diversified.

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada.

BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata.

BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

The Oxymonad Genome Displays Canonical Eukaryotic Complexity in the Absence of a Mitochondrion.

The discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less "reduced" than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe-S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.

Genomic survey maps differences in the molecular complement of vesicle formation machinery between Giardia intestinalis assemblages.

Giardia intestinalis is a globally important microbial pathogen with considerable public health, agricultural, and economic burden. Genome sequencing and comparative analyses have elucidated G. intestinalis to be a taxonomically diverse species consisting of at least eight different sub-types (assemblages A-H) that can infect a great variety of animal hosts, including humans. The best studied of these are assemblages A and B which have a broad host range and have zoonotic transmissibility towards humans where clinical Giardiasis can range from asymptomatic to diarrheal disease. Epidemiological surveys as well as previous molecular investigations have pointed towards critical genomic level differences within numerous molecular pathways and families of parasite virulence factors within assemblage A and B isolates. In this study, we explored the necessary machinery for the formation of vesicles and cargo transport in 89 Canadian isolates of assemblage A and B G. intestinalis. Considerable variability within the molecular complement of the endolysosomal ESCRT protein machinery, adaptor coat protein complexes, and ARF regulatory system have previously been reported. Here, we confirm inter-assemblage, but find no intra-assemblage variation within the trafficking systems examined. This variation includes losses of subunits belonging to the ESCRTIII as well as novel lineage specific duplications in components of the COPII machinery, ARF1, and ARFGEF families (BIG and CYTH). Since differences in disease manifestation between assemblages A and B have been controversially reported, our findings may well have clinical implications and even taxonomic, as the membrane trafficking system underpin parasite survival, pathogenesis, and propagation.

The reduced ARF regulatory system in Giardia intestinalis pre-dates the transition to parasitism in the lineage Fornicata.

Giardia intestinalis is an enteric pathogen with an extremely modified membrane trafficking system, lacking canonical compartments such as the Golgi, endosomes, and intermediate vesicle carriers. By comparison the fornicate relatives of Giardia possess greater endomembrane system complexity. In eukaryotes, the ADP ribosylation factor (ARF) GTPase regulatory system proteins, which consist of the small GTPase ARF1, and its guanine exchange nucleotide factors (GEFs) and GTPase activating proteins (GAPs), coordinate temporal and directional trafficking of cargo vesicles by recognizing and interacting with heterotetrameric coat complexes at pre-Golgi and post-Golgi interfaces. To understand the evolution of this regulatory system across the fornicate lineage, we have performed comparative genomic and phylogenetic analyses of the ARF GTPases, and their regulatory GAPs and GEFs in fornicate genomes and transcriptomes. Prior to our analysis of the fornicates, we first establish that the ARF GAP sub-family ArfGAP with dual PH domains (ADAP) is sparsely distributed but present in at least four eukaryotic supergroups and thus was likely present in the Last Eukaryotic Common Ancestor (LECA). Next, our collective comparative genomic and phylogenetic investigations into the ARF regulatory proteins in fornicates identify a duplication of ARF1 GTPase yielding two paralogues of ARF1F proteins, ancestral to all fornicates and present in all examined isolates of Giardia. However, the ARF GEF and ARF GAP complement is reduced compared with the LECA. This investigation shows that the system was significantly streamlined prior to the fornicate ancestor but was not further reduced concurrent with a transition into a parasitic lifestyle.

Evolution: Parallel Paths to Parasitism in the Apicomplexa.

A new study presents the first comprehensive genome and transcriptome data for an enigmatic group of apicomplexan parasites, the gregarines. The findings provide insights into the early evolution of parasitism in the apicomplexans and illustrate the important contributions of convergent and parallel evolution in the rise of eukaryotic parasites.

Ancient complement and lineage-specific evolution of the Sec7 ARF GEF proteins in eukaryotes.

Guanine nucleotide exchange factors (GEFs) are the initiators of signaling by every regulatory GTPase, which in turn act to regulate a wide array of essential cellular processes. To date, each family of GTPases is activated by distinct families of GEFs. Bidirectional membrane trafficking is regulated by ADP-ribosylation factor (ARF) GTPases and the development throughout eukaryotic evolution of increasingly complex systems of such traffic required the acquisition of a functionally diverse cohort of ARF GEFs to control it. We performed phylogenetic analyses of ARF GEFs in eukaryotes, defined by the presence of the Sec7 domain, and found three subfamilies (BIG, GBF1, and cytohesins) to have been present in the ancestor of all eukaryotes. The four other subfamilies (EFA6/PSD, IQSEC7/BRAG, FBX8, and TBS) are opisthokont, holozoan, metazoan, and alveolate/haptophyte specific, respectively, and each is derived from cytohesins. We also identified a cytohesin-derived subfamily, termed ankyrin repeat-containing cytohesin, that independently evolved in amoebozoans and members of the SAR and haptophyte clades. Building on evolutionary data for the ARF family GTPases and their GTPase--activating proteins allowed the generation of hypotheses about ARF GEF protein function(s) as well as a better understanding of the origins and evolution of cellular complexity in eukaryotes.