The dura mater hosts a rich population of B cell progenitors, but its capacity to sustain B cell development during systemic lymphopenia remains unclear. Using a murine model of chronic Trypanosoma brucei infection, which induces peripheral B cell depletion, we demonstrate that the dura mater maintains intact B cell lymphopoiesis independently of bone marrow and spleen in both male and female mice, in a process involving various chemotactic and pro-survival factors derived from the dura mater stroma. Furthermore, dura mater-derived B cells exhibit a distinct immunoglobulin repertoire that is distinct from the splenic repertoire and is dominated by Ighv1 family members. The immunoglobulins produce locally at the CNS borders are polyreactive and able to recognise both CNS and parasite antigens. Lastly, adoptive transfer of dura mater B cells, or their cognate antibodies, into B cell-deficient mice delays parasitemia onset, whereas splenic B cells from infected hosts fail to confer similar protection. These findings identify, for the first time, the dura mater as a resilient, autonomous B cell lymphopoietic niche that shapes specialized humoral responses during chronic infection, highlighting its potential role in coordinating immune defense when conventional lymphoid organs are compromised.

The efficacy of numerous vector control initiatives is compromised by growing insecticide resistance among disease-transmitting arthropods of agricultural, veterinary, and public health significance. Previous investigations on hematophagous (blood-feeding) arthropod vectors, including mosquitoes, have indicated that ingesting blood containing inhibitors of the second enzyme in the tyrosine metabolism pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD), results in high insect mortality. Building upon this foundation, we evaluated the insecticidal efficacy of the HPPD inhibitor, nitisinone, against susceptible and pyrethroid-resistant strains of three mosquito species: Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. These mosquitoes are vectors of historical diseases such as malaria, emerged diseases such as Dengue and Zika and emerging viral diseases such as the Oropouche and Usutu viruses. We demonstrate, by employing standard screening assays designed to assess the cuticular uptake of mosquitocidal agents, that nitisinone has mosquitocidal activity when blood-fed mosquitoes contact a nitisinone-coated surface. Notably, there is no discernible disparity in susceptibility to nitisinone between an insecticide-susceptible strain of Anopheles gambiae and two strains carrying multiple insecticide-resistance mechanisms. We conclude that the mosquitocidal mode of action of nitisinone differs from any of the current 37 classes of insecticides as none have a mode of action that specifically interferes with blood digestion. By highlighting the efficacy of nitisinone as a contact-based insecticide, our findings support the potential expansion of vector control strategies where nitisinone is incorporated into classic interventions like treated bednets and indoor residual spraying. ### Competing Interest Statement The authors have declared no competing interest.

Background The emergence of insecticide resistance in insects has led researchers to develop new control tools so that historic gains made in reducing disease transmission are not lost. Attractive targeted sugar baits (ATSBs) are a vector control tool being widely trialled to target insects that feed on plant sugars and blood. We designed a field-friendly, economical and more environmentally responsible sugar feeder for maintaining mosquito colonies and screening potential ATSB candidates. Methods We simultaneously tested, in both male and female Anopheles gambiae mosquitoes, the effect of adding three water-soluble medical and food dyes (Allura Red, fluorescein and tartrazine) to the sugar solution to identify those insects that had ingested sugar from the feeder. To test feeder efficacy to deliver a toxic substance, we assessed the killing using boric acid, which kills both male and female mosquitoes when ingested. Using microscopy techniques compatible with fieldwork, including the use of a mobile phone camera, we documented the efficacy and tissue specificity of the dyes on mosquitoes after they were continuously fed dyed sugar solutions. Results The easy-to-construct sugar feeder is an economical option for testing the efficacy of ATSB components on Anopheles gambiae mosquitoes . Allura Red AC was the preferred dye as it has low toxicity to mosquitoes and allows the researcher to quickly visualise the imbibed sugar meal within the abdomen. Feeding 1% fluorescein dye, but not 0.1%, for longer than five days induced systemic dye distribution, where the mosquito’s wing veins, antennae and legs brightly fluoresced when examined by a handheld black light torch (395-400nm emission). Discussion Developing an affordable sugar feeder to maintain insectary-reared insects and test the efficacy of ATSB candidates involves designing a dye-labelled sugar bait station that is of low-toxicity, reusable and easy to construct using components available in low resource settings such as field stations.

Insecticide resistance in disease-transmitting arthropods of agricultural, veterinary, and public health significance poses a significant threat to vector control programs worldwide. Previous studies demonstrated that blood-feeding arthropod vectors experience high mortality when ingesting blood containing inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD), the second enzyme in tyrosine metabolism. This study investigated the mosquitocidal efficacy of HPPD inhibitors from the β-triketone class of herbicides against both susceptible and pyrethroid-resistant strains of three major disease vector species, including mosquitoes that transmit historical diseases such as malaria, reemerging infections such as dengue and Zika, and emerging viral threats such as Oropouche and Usutu viruses. Four HPPD inhibitors (nitisinone, mesotrione, sulcotrione, and tembotrione) were screened using glass plate tarsal bioassays at 125 mg/m2 against bloodfed Anopheles gambiae s.s. Kisumu. Nitisinone was selected for evaluation against susceptible and pyrethroid-resistant strains of An. gambiae s.s. Kisumu, An. gambiae s.l. Tiassalé 13, An. coluzzii VK7 2014, Culex quinquefasciatus Muhezha, and Aedes aegypti New Orleans. Mosquitocidal activity was assessed using glass plate tarsal contact bioassays, topical application assays (0.0001

Background The emergence of insecticide resistance in insects has led researchers to develop new controltools so that historic gains made in reducing disease transmission are notlost. Attractive targeted sugar baits (ATSBs) are a vector control tool beingwidely trialled to target insects that feed on plant sugars and blood. Wedesigned a field-friendly, economical and more environmentally responsible sugarfeeder for maintaining mosquito colonies and screening potential ATSBcandidates. Methods We simultaneously tested, in both male and female Anopheles gambiae mosquitoes, the effectof adding three water-soluble medical and food dyes (Allura Red, fluoresceinand tartrazine) to the sugar solution to identify those insects that hadingested sugar from the feeder. To test feeder efficacy to deliver a toxicsubstance, we assessed the killing using boric acid, which kills both male andfemale mosquitoes when ingested. Using microscopy techniques compatible with fieldwork,including the use of a mobile phone camera, we documented the efficacy andtissue specificity of the dyes on mosquitoes after they were continuously fed dyedsugar solutions. Results The easy-to-construct sugar feeder is an economical option for testing the efficacyof ATSB components on Anopheles gambiae mosquitoes. Allura Red ACwas the preferred dye as it has low toxicity to mosquitoes and allows theresearcher to quickly visualise the imbibed sugar meal within the abdomen. Feeding1% fluorescein dye, but not 0.1%, for longer than five days induced systemicdye distribution, where the mosquito’s wing veins, antennae and legs brightlyfluoresced when examined by a handheld black light torch (395-400nm emission). Discussion Developing an affordable sugar feeder to maintain insectary-reared insects and test theefficacy of ATSB candidates involves designing a dye-labelled sugar baitstation that is of low-toxicity, reusable and easy to construct usingcomponents available in low resource settings such as field stations.

Cutaneous leishmaniasis (CL) is a parasitic vector-borne disease prevalent in 90 countries. Despite its endemicity in Guatemala, key transmission factors are still unknown. To address this, we characterized sand fly populations and Leishmania parasites in a Guatemalan community in 2022. We visited the households of 23 patients with lesions compatible with CL, sampled for sand flies and analysed Leishmania spp. presence in patients’ skin scrapings and in collected sand flies. We collected 93 sand flies, predominantly females including the vector species Nyssomyia ylephiletor, Bichromomyia olmeca and Lutzomyia cruciata. Nyssomyia ylephiletor was the most abundant species indoors. Four Leishmania spp. were identified including L. panamensis, L. guyanensis, L. braziliensis and L. infantum in CL lesions, L. guyanensis complex species (L. guyanensis or L. panamensis) and Leishmania sp. in sand flies. Sand fly species positive for Leishmania spp. were Ny. ylephiletor, Dampfomyia deleoni, Dampfomyia sp. and Brumptomyia sp. Blood-meal analysis revealed human and pig blood in engorged Ny. ylephiletor collected inside and in the proximity of the households. This is the first report of L. guyanensis in Guatemalan patients and provides insights into CL transmission dynamics, suggesting potential indoor transmission, pending more studies.

One approach to interrupting the transmission of insect-borne diseases that is successfully used in veterinary medicine is exploiting the ability of antiparasitic drugs to make vertebrate blood toxic for blood-feeding insects. Recent studies have identified 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme of the tyrosine detoxification pathway, as essential for hematophagous arthropods to digest their blood meals. Such blood-feeding insects include anopheline mosquitoes, which transmit malaria-causing Plasmodium parasites. A US Food and Drug Administration-approved HPPD enzyme inhibitor called nitisinone is a drug used to treat rare human-inherited disorders of the tyrosine pathway. Here, we demonstrate that feeding human blood containing nitisinone to insectary-reared female Anopheles gambiae mosquitoes was mosquitocidal to both young and old mosquitoes as well as insecticide-resistant Anopheles strains. Pharmacokinetic-pharmacodynamic (PK/PD) modeling of nitisinone's dose-response relationship (when administered at the highest recommended doses for adults and children) demonstrated improved efficacy against mosquitoes compared with the gold standard endectocidal drug, ivermectin. Furthermore, blood samples from individuals with alkaptonuria (a rare genetic metabolic disorder in the tyrosine degradation pathway), who were taking a daily low dose of 2 milligrams of nitisinone, were shown to be lethal to mosquitoes. Thus, inhibiting the Anopheles HPPD enzyme with nitisinone warrants further investigation as a complementary intervention for vector control and the prevention of malaria transmission.

Background Tsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed. Methodology/Principle findings Insectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/Significance Our results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.

Abstract Background Cutaneous leishmaniasis (CL) is a parasitic vector borne disease endemic in 90 countries, including Guatemala. Despite the endemicity of the disease in the country, the most important factors that contribute to its transmission remain unknown. To address these knowledge gaps, we characterized the sand fly populations and Leishmania parasites circulating in patients and sand flies in an endemic community in Guatemala. Methods From March to August 2022, we visited the households of 23 patients recently diagnosed with CL to collect sand flies in three environments: indoors, outdoors in animal sheds and in the surrounding forest. The sand fly species were identified using end-point PCR targeting the cytochrome C gene followed by sequencing, and screened for Leishmania DNA by heat-shock protein 70 gene PCR and sequencing. Using the same methodology on lesion tissue smear material from patients, we identified the species of parasites causing infections in the community. Results We collected 93 sand flies (79% females) of at least five species, three of which have been previously associated with Leishmania transmission: Nyssomyia ylephiletor, Bichromomyia olmeca and Lutzomyia cruciata. The highly anthropophilic Ny. ylpehiletor, of which only females were captured, was the most common species (28%) and was primarily collected indoors (73.1%). Four female sand flies were positive for Leishmania DNA, and one of them (Ny. ylephiletor) was captured indoors. The parasite complex L. guyanensis (L. guyanensis and L. panamensis) was found in both sand flies and patients’ samples. In patients we also detected L. braziliensis and a cutaneous form of L. infantum. Three engorged Ny. ylephiletor were screened for blood meal identification, finding humans and domestic pig’s blood. Conclusions This is the first report in Guatemala of the presence of L. guyanensis in patients, and the first characterization of Leishmania infections in humans and sand flies that concur in time and geographical area. The capture of anthropophilic sand flies indoors, some with Leishmania DNA, could suggest indoor transmission of CL, but further studies are needed to confirm our results. These findings are key to local health authorities for decision-making on the appropriate prevention measures, such as the use of insecticide treated bed nets.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T . brucei -infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T . brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate’s skin.

Cutaneous leishmaniasis (CL) is a parasitic vector-borne disease affecting mostly low- and middle-income countries. CL is endemic in Guatemala, where an increase in the number of cases and incidence and a changing disease distribution in the past decade have been reported. Important research was conducted in Guatemala in the 1980s and 1990s to understand the epidemiology of CL and two Leishmania species were identified as the aetiologic agents. Several species of sand flies have been reported, five of which are naturally infected with Leishmania. Clinical trials conducted in the country evaluated different treatments against the disease and provided solid evidence for CL control strategies that are applicable worldwide. More recently, in the 2000s and 2010s, qualitative surveys were conducted to understand community perceptions of the disease and to highlight the challenges and enablers for disease control. However, limited recent data have been generated regarding the current CL situation in Guatemala, and key information necessary for effective disease control, such as incrimination of vectors and reservoirs, is still lacking. This review describes the current state of knowledge of CL in Guatemala, including the main parasite and sand fly species, disease reservoirs, diagnosis and control, as well as the perceptions of communities in endemic regions.

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These N-glycans are predicted to modulate spike binding to the host receptor by stabilizing its open conformation and host immunity evasion. Here, we investigated the essentiality of both the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Ablation of host N-glycosylation using RNA interference or inhibitors, including FDA-approved drugs, reduced the spread of the infection, including that of variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Under these conditions, cells produced fewer virions and some completely lost their infectivity. Furthermore, partial enzymatic deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, we propose protein N-glycosylation as a targetable pathway with clinical potential for treatment of COVID-19. IMPORTANCE The coronavirus SARS-CoV-2 uses its spike surface proteins to infect human cells. Spike proteins are heavily modified with several N-glycans, which are predicted to modulate their function. In this work, we show that interfering with either the synthesis or attachment of spike N-glycans significantly reduces the spread of SARS-CoV-2 infection in vitro, including that of several variants. As new SARS-CoV-2 variants, with various degrees of resistance against current vaccines, are likely to continue appearing, halting virus glycosylation using repurposed human drugs could result in a complementary strategy to reducing the spread of COVID-19 worldwide.

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1FoATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

34 SARS-CoV-2 extensively N-glycosylates its surface spike (S) proteins. This post35 translational modification is essential to modulate protein conformation and host cell 36 invasion. Each S monomer can be modified with up to 22 N-glycans. To meet the high 37 demand of protein glycosylation during virus replication, SARS-CoV-2 upregulates the 38 expression of host N-glycosylation genes. Although a substantial amount of detail is 39 known about the structure of S protein N-glycans, the role of N-glycosylation in SARS40 CoV-2 infection remains largely undetermined. Here, we investigated the essentiality 41 of the host N-glycosylation pathway and viral N-glycans for SARS-CoV-2 infection. 42 When either monkey or human cells were preincubated with glycosylation inhibitors, 43 including FDA-approved iminosugars, virus infection was significantly reduced. This 44 infection phenotype was confirmed after RNAi knockdown of several glycosylation 45 genes. In addition, enzymatic deglycosylation of whole viral particles confirmed that 46 accessible oligosaccharides on the SARS-CoV-2 surface are essential for host cell 47 infection. Altogether, we show evidence that the normal functioning of the host N48 glycosylation machinery is essential not only for SARS-CoV-2 to infect, but also to 49 produce new functional virions. These findings open the door for developing new 50 approaches targeting N-glycosylation against COVID-19. 51 52

SARS-CoV-2 extensively N -glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These sugars are predicted to help mediate spike binding to the host receptor by stabilizing its ‘open’ conformation and evading host immunity. Here, we investigated both the essentiality of the host N -glycosylation pathway and SARS-CoV-2 N -glycans for infection. Inhibition of host N -glycosylation using RNAi or FDA-approved drugs reduced virus infectivity, including that of several variants. Under these conditions, cells produced less virions and some completely lost their infectivity. Furthermore, partial deglycosylation of intact virions showed that surface-exposed N -glycans are critical for cell invasion. Altogether, spike N -glycosylation is a targetable pathway with clinical potential for treatment or prevention of COVID-19.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids. Introduction The protozoan parasites of the Trypanosoma brucei group are the causative agents of human and animal African trypanosomiasis. Bloodstream form T. brucei is ingested by the tsetse fly vector and differentiates into procyclic form parasites to colonize the tsetse midgut. To then infect a new mammalian host, T. brucei undergoes a series of differentiations that allows it to colonize the fly salivary gland and to be transferred to a new host during a subsequent blood meal (Matthews, 2005). The surface coat of the bloodstream form is characterized by the GPI-anchored, N-glycosylated and occasionally O-glycosylated variant surface glycoprotein (VSG) (Cross, 1996; Mehlert et al., 1998; Pays and Nolan, 1998; Pinger et al., 2018; Schwede and Carrington, 2010), while procyclic cells express a family of GPI-anchored proteins called procyclins (Richardson et al., 1988; Roditi et al., 1998; Treumann et al., 1997; Vassella et al., 2001), free glycoinositolphospholipids (Nagamune et al., 2004; Roper et al., 2005; Vassella et al., 2003), and a high molecular weight glycoconjugate complex (Güther et al., 2009). The importance of glycoproteins to parasite survival and infectivity has led to the investigation of enzymes for GPI anchor biosynthesis (Chang et al., 2002; Nagamune et al., 2000; Smith et al., 2004; Urbaniak et al., 2014; Urbaniak et al., 2008) and nucleotide sugar biosynthesis (Bandini et al., 2012; Denton et al., 2010; Kuettel et al., 2012; Sampaio Guther et al., 2021; Marino et al., 2010; Marino et al., 2011; Roper et al., 2002; Roper et al., 2005; Stokes et al., 2008; Shaw et al., 2003; Turnock et al., 2007; Urbaniak et al., 2006a; Urbaniak et al., 2006b; Urbaniak et al., 2013; Zmuda et al., 2019) as potential therapeutic targets. Nucleotide sugars are used as glycosyl donors in many glycosylation reactions. GDP-fucose (GDP-Fuc) was identified in the nucleotide sugar pools of T. brucei, Trypanosoma cruzi, and Leishmania major (Turnock and Ferguson, 2007), and its biosynthesis is essential for parasite growth in procyclic and bloodstream form T. brucei (Turnock et al., 2007) and in L. major promastigotes (Guo et al., 2017). Interestingly, T. brucei and L. major use different pathways to synthesize GDP-Fuc. T. brucei utilizes the de novo pathway in which GDP-Fuc is synthesized from GDP-mannose via GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) (Sampaio Guther et al., 2021; Turnock et al., 2007; Turnock et al., 2007). Conversely, L. major has two related bifunctional D-arabinose/L-fucose kinase/pyrophosphorylase, AFKP80 and FKP40, that synthesize GDP-Fuc from free fucose (Guo et al., 2017). Despite the aforementioned essentialities for GDP-Fuc in T. brucei and L. major, the only structurally defined fucose-containing oligosaccharide in trypanosomatids is the low-abundance Ser/Thr-phosphodiester-linked glycan on T. cruzi gp72, a glycoprotein that has been implicated in flagellar attachment (Allen et al., 2013; Cooper et al., 1993; Ferguson et al., 1983; Haynes et al., 1996). Fucosyltransferases (FUTs) catalyse the transfer of fucose from GDP-Fuc to glycan and protein acceptors and are classified into two superfamilies (Coutinho et al., 2003; Lombard et al., 2014). One superfamily contains all α1,3/α1,4-FUTs (carbohydrate active enzyme, CAZy, family GT10) and the other contains all α1,2-, α1,6-, and protein O-fucosyltransferases (GT11, GT23, GT37, GT56, GT65, GT68, and GT74; Martinez-Duncker, 2003). In eukaryotes, FUTs are generally either type II transmembrane Golgi proteins or ER-resident enzymes (POFUT1 and POFUT2) (Breton et al., 1998), but two exceptions have been described: (i) PgtA, a cytoplasmic bifunctional β1,3-galactosyltransferase α1,2-FUT found in Dictyostelium discoideum and Toxoplasma gondii (Rahman et al., 2016; Van Der Wel et al., 2002) that is part of an oxygen-sensitive glycosylation pathway that attaches a pentasaccharide to the Skp1-containing ubiquitin ligase complex (West et al., 2010); and (ii) SPINDLY, a protein O-fucosyltransferase that modifies nuclear proteins in Arabidopsis thaliana and T. gondii (Gas-Pascual et al., 2019; Zentella et al., 2017). T. brucei and other kinetoplastids contain a single mitochondrion. In the bloodstream form of the parasite, this organelle has a tubular structure, while in the procyclic form it is organized in a complex network with numerous cristae, reflecting the absence and presence, respectively, of oxidative phosphorylation (Matthews, 2005; Priest and Hajduk, 1994). The parasite mitochondrion is further characterized by a disc-shaped DNA network called the kinetoplast (Jensen and Englund, 2012) that is physically linked with the flagellum basal body (Ogbadoyi et al., 2003; Povelones, 2014). While secretory pathway and nuclear/cytosolic glycosylation systems have been studied extensively, little is known about glycosylation within mitochondria. A glycoproteomic approach in yeast revealed several mitochondrial glycoproteins (Kung et al., 2009), but it was not determined whether these were imported from the secretory pathway or glycosylated within the mitochondria by as yet unknown glycosyltransferases. The only characterized example of a mitochondrial glycosyltransferase is the mitochondrial isoform of mammalian O-GlcNAc transferase (OGT). O-GlcNAcylation is a cycling modification, involved in signalling, in which OGT adds GlcNAc to Ser/Thr residues and O-GlcNAcase (OGA) removes it (Bond and Hanover, 2015). Studies have shown that both mitochondrial OGT (mOGT) and OGA are present and active in mammalian mitochondria and putative mitochondrial targets have been identified (Banerjee et al., 2015; Sacoman et al., 2017). Further, a mammalian mitochondrial UDP-GlcNAc transporter associated with mitochondrial O-GlcNAcylation has been described (Banerjee et al., 2015). However, orthologues of OGT and OGA genes are not present in kinetoplastids. Here, we report on a gene (TbFUT1) encoding a mitochondrial α-1,2-fucosyltransferase protein (TbFUT1) in T. brucei that is essential to parasite survival. Similar results were obtained in the related trypanosomatid parasite L. major (Guo et al., 2021), extending this unexpected finding across the trypanosomatid protozoans. Results Identification, cloning, and sequence analysis of TbFUT1 The CAZy database lists eight distinct FUT families (see Introduction) (Lombard et al., 2014). One or more sequences from each family were selected for BLASTp searches of the predicted proteins from the T. brucei, T. cruzi, and L. major genomes (Supplementary file 1). Strikingly, only one putative fucosyltransferase gene (TbFUT1) was identified in the T. brucei genome (GeneDB ID: Tb927.9.3600) belonging to the GT11 family, which is comprised almost exclusively of α-1,2-FUTs (Coutinho et al., 2003; Zhang et al., 2010). Homologues of TbFUT1 were also found in the T. cruzi and L. major genomes and, unlike T. brucei, T. cruzi, and L. major, also encode for GT10 FUT genes (Supplementary file 1). Finally, Leishmania spp. express a family of α-1,2-arabinopyranosyltransferases (SCA1/2/L, CAZy family GT79) that decorate phosphoglycan side chains and have been suggested to act as FUTs in presence of excess fucose (Guo et al., 2021). The TbFUT1 predicted amino acid sequence shows relatively low sequence identity to previously characterized GT11 FUTs, for example, Helicobacter pylori (26%) or human FUT2 (21%) (Kelly et al., 1995; Wang et al., 1999). Nevertheless, conserved motifs characteristic of this family can be identified (Figure 1A; Li et al., 2008; Martinez-Duncker, 2003). Motif I (aa 153–159) is shared with α-1,6-FUTs and has been implicated in the binding of GDP-Fuc (Takahashi et al., 2000), whereas no clear functions have yet been assigned to motifs II, III, and IV (aa 197–207, 265–273, and 13–18, respectively). Notably, TbFUT1 lacks an identifiable N-terminal signal peptide and has only a low-confidence prediction for a type II membrane protein N-terminal transmembrane domain (0.34108 on TMHMM-2.0 for residues 6–28). These two features would be expected of a typical Golgi-localized FUT (Breton et al., 1998; Figure 1A). Indeed, further analysis of the TbFUT1 predicted amino acid sequence using PSort II (43.5% mitochondrion, 4.3% secretory pathway, 26.1% cytosolic, Horton and Nakai, 1997), Target P (0.428 mitochondrial, 0.064 secretory, 0.508 other, Emanuelsson et al., 2000), and Mitoprot (0.5774 probability of mitochondrial localization, Claros and Vincens, 1996) rated the localization as most likely mitochondrial and identified a putative mitochondrial targeting motif (M/L … RR) with RR at sequence positions 30 and 31. Conservation of this eukaryotic targeting motif has been previously shown for other parasite mitochondrial proteins (Krnáčová et al., 2012; Long et al., 2008). Figure 1 Download asset Open asset Amino acid sequence and phylogenetic analyses of TbFUT1. (A) Sequence alignment of TbFUT1 and other GT11 family fucosyltransferases (FUTs) shows that TbFUT1 (Tb927.9.3600) lacks a conventional N-terminal type 2 membrane protein transmembrane domain (TM, grey box) but contains conserved motifs I–IV (black boxes). A putative TbFUT1 TM (dashed box) overlaps with motif IV but does not align with the eukaryotic FUT TM and is most likely part of a cleavable N-terminal mitochondrial targeting sequence (residues 1–31 with cleavage at R30–R31). Sequences used in the alignment: T. cruzi (TcCLB.506893.90), L. major (LmjF01.0100), H. pylori (AAC99764), Homo sapiens FUT2 (AAC24453), and Mus musculus FUT2 (AAF45146). (B) A selection of known and predicted fucosyltransferase protein sequences was aligned using ClustalΩ (Sievers et al., 2011), and the unrooted phylogram shown was generated by iTOL (itol.embl.de) (Letunic and Bork, 2016). Single homologues of the GT11 TbFUT1 were found in all the kinetoplastids (marked in green) and, collectively, these sequences form a clade distant from other fucosyltransferase sequences, including the other kinetoplastid GT10 fucosyltransferases (marked by a dashed line). No TbFUT1 homologues were found in apicomplexan parasites or in Euglena. Additional BLASTp searches showed that there is generally a single TbFUT1 gene homologue in each kinetoplastid species, and a phylogram of FUT sequences indicates that TbFUT1 homologues form a distinct clade closest to bacterial α-1,2-FUT (Figure 1B). Recombinant expression of TbFUT1 The TbFUT1 ORF was amplified from T. brucei 427 genomic DNA and cloned in the pGEX6P1 expression vector. The resulting construct (pGEX6P1-GST-PP-TbFUT1) encoded for the TbFUT1 ORF with a glutathione-S-transferase (GST) tag at its N-terminus and a PreScission Protease (PP) cleavage site between the two protein-encoding sequences. Sequencing confirmed what was subsequently deposited at TriTrypDB (Tb427_090021700) and identified two amino acid differences between TbFUT1 in the 927 and 427 strains (L185V and T232A). The pGEX6P1-GST-PP-TbFUT1 construct was expressed in Escherichia coli and the fusion protein purified as described in Materials and methods. The identities of the two higher molecular weight bands (Figure 2—figure supplement 1, lane 8) were determined by peptide mass fingerprinting. The most abundant band was identified as TbFUT1, while the fainter band was identified as a subunit of the E. coli GroEL chaperonin complex. The apparent molecular weight of GST-PP-TbFUT1 chimeric protein (57 kDa) was consistent with the predicted theoretical molecular weight (58.1 kDa). Recombinant TbFUT1 is active in vitro The activity of recombinantly expressed GST-TbFUT1 fusion protein was tested by incubation with GDP-[3H]Fuc, as a donor, and a panel of commercially available mono- to octasaccharides (Table 1) selected from the literature as possible α-1,2-FUT substrates (Li et al., 2008; Wang et al., 1999; Zhang et al., 2010). The effectiveness of each acceptor was evaluated based on the presence/absence and intensities of the TLC bands corresponding to the radiolabelled reaction products (Figure 2 and Table 1). GST-TbFUT1 showed best activity with Galβ1,3GlcNAc (LNB) (Figure 2, lane 2) and its β-O-methyl glycoside (Figure 2, lane 21). Other larger oligosaccharides containing Galβ1,3GlcNAcβ1-O-R as a terminal motif (LNT and LNH) were also good acceptors (Figure 2, lanes 11 and 15), with the exception of iLNO (Figure 2, lane 13). Lactose was also recognized (Figure 2, lane 1), while LacNAc and the LacNAc-terminating branched hexasaccharide LNnH were weak acceptors (Figure 2, lanes 3 and 10). Interestingly, TbFUT1 was also able to transfer fucose to 3′-fucosyllactose, albeit inefficiently (Figure 2, lane 16), whereas no transfer could be seen to Galβ1,6GlcNAc (Figure 2, lane 17) or to free Gal or β-Gal-O-methyl (Figure 2, lanes 9 and 20). As expected, no products were observed when acceptor oligosaccharides were omitted from the reaction (Figure 2, lane 4). To confirm the detected activities were specific to the recombinant GST-TbFUT1, and not due to some co-purifying endogenous E. coli contaminant, the assay was also performed using material prepared from E. coli expressing the empty pGEX6P1 vector. No transfer of radiolabelled fucose could be observed under these conditions (Figure 2, lanes 5–7). Table 1 Acceptor substrates and semi-quantitative fucosyltransferase activities. TbFUT1 activityLane of Figure 1Abbreviations*NameStructure+++2, 6LNBLacto-N-bioseGalβ1,3GlcNAc+++21LNB-OMeLacto-N-biose-O-methylGalβ1,3GlcNAcβ-OMe++11LNTLacto-N-tetraoseGalβ1,3GlcNAcβ1,3Galβ1,4Glc++14LNHLacto-N-hexaoseGalβ1,3GlcNAcβ1,3(Galβ1,4GlacNAcβ1,6)Galβ1,4Glc++1,5LacLactoseGalβ1,4Glc+13iLNOIso-lacto-N-octaoseGalβ1,3GlcNAcβ1,3(Galβ1,3GlcNAcβ1,3Galβ1,4GlcNAcβ1,6)Galβ1,4Glc+3,7LacNAcN-acetyllactosamineGalβ1,4GlcNAcN+10LNnHLacto-N-neohexaoseGalβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc+12LNnTLacto-N-neotetraoseGalβ1,4GlcNAcβ1,3Galβ1,4Glc+163′-FL3′-FucosyllactoseGalβ1,4(Fucα1,3)Glc–9β-Galβ-Galactoseβ-Gal–15GNGβ1,6-Galactosyl-N-acetyl-glucosamineGalβ1,6GlcNAc–171,6 GBβ1,6-GalactobioseGalβ1,6Gal–181,4 GBGalabioseGalα1,4Gal–19LB2TSLinear B2 trisaccharideGalα1,3Galβ1,4GlcNAc–20β-Gal-OMeβ-Galactose-O-methylβ-Gal-OMe–222'-FL2'-FucosyllactoseFucα1,2Galβ1,4Glc * The relative efficiencies of the acceptors as TbFUT1 substrates (+++, ++, +, and –) are based on visual inspection of the intensities of the products bands in Figure 2. Figure 2 with 2 supplements see all Download asset Open asset Recombinant GST-TbFUT1 transfers [3 H]Fuc to a variety of sugar acceptors. Each assay used 2 μg of purified GST-TbFUT1, GDP-[3H]Fuc, and 1 mM of acceptor. Reaction products were desalted and separated by silica High Performance Thin Layer Chromatography (HPTLC) and detected by fluorography. LNB, LNB-OMe, and LNB-terminating structures were the best acceptors tested. The acceptor abbreviations above each lane are defined in Table 1. -ctrl: negative control reaction without acceptor (lane 4) or with buffer alone (lane 8). A band with the same mobility as free fucose was observed in all assay reactions and was considerably stronger in the presence of the GST-TbFUT1 preparation (Figure 2, lanes 1–4 and 9–22) than when GDP-[3H]Fuc was incubated with reaction buffer alone (Figure 2, lane 8) or in the presence of the material purified from the E. coli cells transformed with the empty vector (Figure 2, lanes 5–7). These data suggest that TbFUT1 has a significant propensity to transfer Fuc to water. Interestingly, one of the substrates (LNB-O-Me; Galβ1,3GlcNAcβ1-O-methyl) suppressed the amount of free Fuc produced in the reaction (Figure 2, lane 21), suggesting that this glycan may bind more tightly to the TbFUT1 acceptor site than the other oligosaccharides tested and thus prevent the transfer of Fuc from GDP-Fuc to water. In vitro high sugar nucleotide hydrolysis activity has been previously described for at least one member of the GT11 family (Zhang et al., 2010). Inverting α-1,2 and α-1,6-FUTs do not usually require divalent cations for their activity (Beyer and Hill, 1980; Kamińska et al., 2003; Li et al., 2008; Pettit et al., 2010). To study the divalent cation dependence of TbFUT1, the assay was repeated in buffer without divalent cations or containing EDTA. No change in activity was observed in either case, indicating TbFUT1 does not require divalent cations for its activity (Figure 2—figure supplement 2). Finally, given the propensity for TbFUT1 to transfer [3H]Fuc from GDP-[3H]Fuc to water, producing free [3H]Fuc and presumably GDP, we set up a GDP-Glo assay similar to that used for LmjFUT1 (Guo et al., 2021) to monitor the turnover by recombinant TbFUT1 of non-radioactive GDP-Fuc to GDP (see Materials and methods). In this assay, we could see TbFUT1-dependent turnover of GDP-Fuc to GDP in the absence of acceptor substrate (70 ± 2 pmol) and the stimulation of turnover in the presence of LNB acceptor substrate (265 ± 13 pmol), consistent with the results in the radiometric assay. Under the same conditions, there was no detectible turnover of either GDP-Man or GDP-Glc, showing that TbFUT1 is specific, or at least highly selective, for GPD-Fuc as donor substrate. Characterization of the TbFUT1 reaction product The glycan reaction products were structurally characterized to determine the anomeric and stereochemical specificity of TbFUT1. Initially, we performed exoglycosidase and/or acid treatment of the radiolabelled reaction products (recovered by preparative TLC) utilizing Lac, LacNAc, and LNB as substrates. The tritium label ran with the same mobility as authentic Fuc after acid hydrolysis of all three products (Figure 3—figure supplement 1A,C) and after Xanthomonas manihotis α-1,2-fucosidase digestion of the Lac and LNB products (Figure 3—figure supplement 1B,C). These data suggest that [3H]Fuc was transferred in α1,2 linkage to the acceptor disaccharides. To obtain direct evidence, we performed a large-scale activity assay using LNB-O-Me as an acceptor and purified the reaction product by normal phase HPLC. Fractions containing the putative fucosylated trisaccharide product (Figure 3—figure supplement 2) were pooled and analysed for their neutral monosaccharide content, which showed the presence of Fuc, Gal, and GlcNAc. The purified reaction products were permethylated and analysed by electrospray ionisation mass spectrometry (ESI-MS) (Figure 3A), which confirmed that the main product was a trisaccharide of composition dHex1Hex1HexNAc1. The MS/MS spectrum was also consistent with the dHex residue being attached to the Hex, rather than HexNAc, residue (Figure 3B). Subsequently, partially methylated alditol acetates (PMAAs) were generated from the purified trisaccharide product and analysed by gas chromatography coupled to mass spectrometry (GC-MS). This analysis identified derivatives consistent with the presence of non-reducing terminal-Fuc, 2-O-substituted Gal, and 3-O-substituted GlcNAc (Figure 4—figure supplement 1 and Table 2), consistent with Fuc being linked to position 2 of Gal. The GC-MS methylation linkage analysis also revealed a trace of 2-O-substituted Fuc in the sample, which, together with the observation that 3′-FL, can act as a weak substrate (Figure 2, lane 18, and Table 1), may suggest that TbFUT1 can also form Fucα1,2Fuc linkages. Figure 3 with 2 supplements see all Download asset Open asset ESI-MS and ESI-MS/MS of TbFUT1 in vitro reaction product. (A) ESI-MS of the purified and permethylated reaction product. The ion at m/z 692.5 is consistent with the [M + Na]+ ion of a permethylated trisaccharide of composition dHex1Hex1HexNAc1. Some of the unmodified acceptor (Hex1HexNAc1) was also observed (m/z 518.4). (B) MS/MS product ion spectrum of m/z 692.5. The collision-induced fragmentation pattern indicated that the dHex (Fuc) residue was linked to the Hex (Gal) and not to the HexNAc (GlcNAc) residue. Table 2 Partially methylated alditol acetates (PMAAs) derivatives identified by GC-MS methylation linkage analysis of the purified TbFUT1 reaction product. PMAA derivativeRT (min)Origin4,6-di-O-methyl-1,3,5-tri-O-acetyl-(1–2H)- 2-N-methylacetamidoglucosaminitol24.63-O-substituted GlcNAc2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl-(1–2H)-galactitol16.7Non-reducing terminal Gal3,4,6-tri-O-methyl-1,2,5-tri-O-acetyl-(1–2H)-galactitol18.62-O-substituted Gal2,3,4-tri-O-methyl-1,5-di-O-acetyl-(1–2H)-fucitol14.1Non-reducing terminal Fuc3,4-di-O-methyl-1,2,5-tri-O-acetyl-(1–2H)-fucitol15.92-O-substituted Fuc RT: retention time. The purified TbFUT1 reaction product was also exchanged into deuterated water (2H2O) and analysed by one-dimensional 1H-NMR and two-dimensional 1H-ROESY (Rotating frame Overhouser Effect SpectroscopY). The 1H-NMR spectrum showed a doublet at about 5.1 ppm, consistent with the signal from the proton on the anomeric carbon (H1) of an α-Fuc residue (Figure 4A). Figure 4 with 1 supplement see all Download asset Open asset 1H-NMR and 1H-ROESY spectra of the TbFUT1 reaction product. (A) One-dimensional 1H-NMR spectrum. The arrow points to the α-Fuc H1 doublet. (B) Enlargement of the 3.2–5.1 ppm region of the two-dimensional 1H-ROESY spectrum. (a) indicates the crosspeak resulting from a through-space connectivity between α-Fuc H1 and β-Gal H2. A characteristic doublet for the anomeric proton of a β-Gal residue was also observed at 4.5 ppm. In the 1H-ROESY spectrum, a cross-peak (labelled a) could be observed indicating a through-space connectivity between the H1 of α-Fuc and the H2 of a β-Gal, consistent with a Fucα1,2Gal linkage in the TbFUT1 reaction product (Figure 4B). The chemical shifts that could be clearly assigned by either one-dimensional 1H-NMR or two-dimensional 1H-ROESY are listed in Table 3. Table 3 1H-NMR and 1H-ROESY chemical shift assignments for the purified TbFUT1 reaction product. ResidueH1H2H3H4H5H6/6'NAcαFuc5.05 (J = 4 Hz)3.573.673.634.21.1βGal4.53.453.553.89NDNDβGlcNAcND3.63ND3.4ND3.78/3.892.1 J: coupling constant; ND: chemical shift could not be clearly assigned. Taken together, these data unambiguously define the structure of the TbFUT1 reaction product with GDP-Fuc and LNB-O-Me as Fucα1,2Galβ1,3GlcNAcβ1-O-CH3 which, in turn, defines TbFUT1 as having a GDP-Fuc: β-Gal α-1,2-fucosyltransferase activity with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Generation of TbFUT1 conditional null mutants in procyclic and bloodstream form T. brucei Semi-quantitative RT-PCR showed that TbFUT1 mRNA was present in both bloodstream form and procyclic form T. brucei (data not shown). We therefore sought to explore TbFUT1 function in both lifecycle stages by creating TbFUT1 conditional null mutants. The strategies used to generate the mutants are described in Figure 5. The creation of these mutants was possible because genome assembly indicated TbFUT1 to be present as a single copy per haploid genome, and Southern blot analysis using a TbFUT1 probe was consistent with this prediction (Figure 5—figure supplement 1). In procyclic cells (Figure 5, left panel), the first TbFUT1 allele was replaced by homologous recombination with linear DNA containing the puromycin resistance gene (PAC) flanked by about 500 bp of the TbFUT1 5′- and 3′-UTRs. After selection with puromycin, an ectopic copy of TbFUT1, under the control of a tetracycline-inducible promoter, was introduced in the ribosomal DNA (rDNA) locus using phleomycin selection. Following induction with tetracycline, the second allele was replaced with the BSD gene by homologous recombination, generating the final procyclic form ΔTbFUT1::PAC/TbFUT1Ti/ΔTbFUT1::BSD conditional null mutant cell line (PCF TbFUT1 cKO). In bloodstream form cells (Figure 5, middle panel), an ectopic copy of TbFUT1 carrying a C-terminal MYC3 epitope tag under the control of a tetracycline-inducible promoter was first introduced into the ribosomal DNA (rDNA) locus using phleomycin selection. Following cloning and induction with tetracycline, the first TbFUT1 allele was then targeted for homologous recombination with linear DNA containing the hygromycin resistance gene (HYG) flanked by about 1200 bp of the TbFUT1 5′- and 3′-UTRs. After selection with hygromycin, Southern blotting revealed that gene conversion had taken place and that both TbFUT1 alleles had been replaced by HYG yielding a bloodstream form TbFUT1-MYC3Ti/ΔTbFUT1::HYG/ΔTbFUT1::HYG conditional null mutant cell line (BSF TbFUT1-MYC3 cKO). Southern blotting data confirming the genotypes of these mutants are shown in Figure 5—figure supplement 1. The BSF cell line was also used to generate a TbFUT1Ti/ΔTbFUT1::HYG/ΔTbFUT1::HYG conditional null mutant cell line by in situ homologous recombination of the tetracycline inducible TbFUT1-MYC3 copy, converting it to an untagged TbFUT1 gene and generating BSF TbFUT1 cKO (Figure 5, right panel). Figure 5 with 1 supplement see all Download asset Open asset Cloning strategies for the creation of the TbFUT1 conditional null mutants. Left panel: To create the procyclic form conditional null mutant (PCF TbFUT1 cKO), the first TbFUT1 allele was replaced by PAC, an ectopic tetracycline-inducible copy of the TbFUT1 gene was introduced into the ribosomal DNA locus, and the second TbFUT1 allele was replaced by BSD. Middle panel: To create the bloodstream form conditional null mutant (BSF TbFUT1-MYC3 cKO), an ectopic tetracycline-inducible copy of the TbFUT1 gene with a MYC3 tag was first introduced into the ribosomal DNA locus. Both TbFUT1 alleles were subsequently replaced by HYG through homologous recombination followed by gene conversion. Right panel: To create the untagged bloodstream form cKO (BSF TbFUT1 cKO), the BSF TbFUT1-MYC3 cKO mutant (middle panel) was modified by homologous recombination with a construct that removed the C-terminal MYC3 tag under PAC selection. IGR: intergenic region. TbFUT1 is essential to procyclic and bloodstream form T. brucei Procyclic and bloodstream form TbFUT1 conditional null mutants were grown under permissive (plus tetracycline) or non-permissive (minus tetracycline) conditions. The PCF TbFUT1 cKO cells grown under non-permissive conditions showed a clear reduction in the rate of cell growth after 6 days, eventually dying after 15 days (Figure 6A). Figure 6 with 4 supplements see all Download asset Open asset TbFUT1 is essential for procyclic and bloodstream form cell growth in vitro. The cell numbers (± standard deviation) for TbFUT1 cKO under permissive (plus tetracycline, dotted line) and non-permissive (minus tetracycline, solid line) conditions are shown for three procyclic (A) and bloodstream form (C) clones, as well as for three bloodstream clones carrying a tetracycline-inducible ectopic TbFUT1 gene with a C-terminal MYC3 tag (B). For each clone procyclic form clone, two biological repeats were analysed and three for bloodstream form clones. (D, E) Corresponding TbFUT1 mRNA levels were determined by northern blots. Alpha-tubulin (TUB) was used as a loading control. (F) TbFUT1-MYC3 and untagged TbFUT1 are detected by western blot analysis in the respective bloodstream form cKO cell lines under permissive conditions (+Tet). The left panel shows an anti-MYC (αMYC) blot and the right panel an anti-recombinant TbFUT1 antibody (αFUT1) blot. Membranes were stained with Ponceau S (PS) to ensure equal loading. The BSF TbFUT1 cKO cells grew like wild-type cells under permissive conditions, whether or not the expressed TbFUT1 had a C-terminal MYC3 tag, and under non-permissive conditions also showed a clear reduction in the rate of cell growth after 2–4 days, dying after 3–5 days (Figure 6B,C). These growth phenotypes are very similar to those described for procyclic and bloodstream form TbGMD conditional null mutants that cannot synthesize GDP-Fuc under non-permissive conditions (Figure 6—figure supplement 1; Turnock et al., 2007). This is consistent with the hypothesis that TbFUT1 may be the only enzyme that utilizes GDP-Fuc, or at least that it is the only FUT transferring fucose to essential acceptors. Further evidence that TbFUT1 is essential for procyclic and bloodstream form growth was obtained from northern blots (Figure 6D,E). These show that TbFUT1 mRNA levels are undetectable for several days after the removal of tetracycline, but that growth resumes only when some cells escape tetracycline control after about 29 days