The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body via the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions.

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As the protozoan parasite Toxoplasma gondii disseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution. Toxoplasma expresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created a Toxoplasma strain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O2, TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O2 tensions as well as resistance to reactive oxygen species. In vivo, reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O2 levels, in contrast to TgPHYa, which supports the parasite at low O2. IMPORTANCE Because oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O2-sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O2-sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasite Toxoplasma gondii, we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology of Toxoplasma.

Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites.

20 21Hypoxia Inducible Factor-1 is a metazoan heterodimeric transcription factor that senses 22 changes in O 2 levels. HIF-1α subunit abundance is post-translationally regulated by 23 prolyl-hydroxylase domain enzymes (PHDs), which use molecular O 2 and α-24 ketoglutarate to hydroxylate two prolyl-residues in HIF-1α. Three PHDs have been 25 identified and PHD2 is the most critical regulator of HIF-1α. HIF-1α can also be 26 activated independently of hypoxia and in some cases this is due to changes in PHD2 27 abundance through poorly understood mechanisms. Previously, we reported that under 28 O 2 -replete conditions that the intracellular parasite Toxoplasma gondii activates HIF-1 29 by reducing PHD2 protein abundance. Here, we demonstrate that Toxoplasma 30 regulates PHD2 through a multistep process. First, PHD2 is a nucleocytoplasmic 31 protein and Toxoplasma induces PHD2 cytoplasmic accumulation to separate it from 32 nuclear HIF-1α. PHD2 is then degraded by lysosomes independently of the major 33 autophagic processes, macroautophagy or chaperone-mediated autophagy. Rather, 34

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