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[Background] The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely., [Results] Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system., [Conclusions] In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Cellular DNA is inherently unstable, subject to both spontaneous hydrolysis and attack by a range of exogenous and endogenous chemicals as well as physical agents such as ionizing and ultraviolet radiation. For parasitic protists, where an inoculum of infectious parasites is typically small and natural infections are often chronic with low parasitemia, they are also vulnerable to DNA damaging agents arising from innate immune defenses. The majority of DNA damage consists of relatively minor changes to the primary structure of the DNA, such as base deamination, oxidation, or alkylation and scission of the phosphodiester backbone. Yet these small changes can have serious consequences, often being mutagenic or cytotoxic. Cells have therefore evolved efficient mechanisms to repair such damage, with base excision and single strand break repair playing the primary role here. In this chapter we describe a method for analyzing the activity from cell extracts of various enzymes involved in the base excision and single strand break repair pathways of trypanosomatid parasites.

Mass spectrometry based proteomics allows for the identification and quantification of protein and phosphorylation site abundance on a proteome wide scale. Here we describe the metabolic labeling of cultured Trypanosoma brucei cells in either the bloodstream or procyclic life cycle stage using stable isotope labeling of amino acids in cell culture (SILAC), and the production of samples suitable for analysis by liquid chromatography tandem mass spectrometry. The protocols require little specialist equipment, and they typically enable quantification of over 4500 proteins and 9000 phosphorylation sites.

[Background]: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts., [Results]: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids., [Conclusions]: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the ‘shopping bag’ hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, TbFOPL and TbOFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei, showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, TbFOPL, affects neither axoneme nor flagellum elongation. Instead, TbFOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar TbFOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, TbFOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for TbFOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of TbFOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.