Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.


Introduction
The process of protein N-glycosylation in T. brucei has common and unique features compared to other eukaryotes, reviewed in [1] and summarised in (Fig 1).One unique feature is the synthesis of exceptionally large neutral poly-N-acetyllactosamine (pNAL) containing complex N-glycans by bloodstream form (BSF) trypomastigotes [2], the function(s) of which is/are unclear.
Protein N-linked glycosylation in eukaryotes requires the translocation of nascent proteins into the endoplasmic reticulum (ER), where Asn-Xaa-Ser/Thr sequons are N-glycosylated by an oligosaccharyltransferase (OST) which generally transfers Glc3Man9GlcNAc2 from the lipidlinked oligosaccharide (LLO) Glc3Man9GlcNAc2-PP-dolichol to the sidechain nitrogen of the Asn residue.Further, eukaryotic OSTs are generally hetero-oligomeric complexes composed of a catalytic STT3 subunit and up to eight additional subunits.However, in common with other trypanosomatid parasites [3,4], T. brucei OSTs only contain STT3 subunits.The T. brucei genome encodes three complete STT3 genes (TbSTT3A, TbSTT3B and TbSTT3C) and their protein products exhibit different specificities for their donor and acceptor substrates [5][6][7].
Thus, in BSF trypanosomes TbSTT3A acts first and transfers biantennary Man5GlcNAc2 from the lipid-linked oligosaccharide (LLO) Man5GlcNAc2-PP-dolichol to sequons in acidic environments whilst TbSTT3B transfers triantennary Man9GlcNAc2 from LLO Man9GlcNAc2-PPdolichol to remaining unoccupied sequons.The role of TbSTT3C is unclear as it is not well expressed in BSF or insect stage (procyclic) T. brucei, but from its expression in yeast it appears to have the LLO donor specificity of TbSTT3B with the acceptor sequon environment specificity of TbSTT3A [8,9].Not only is protein N-glycosylation in T. brucei unusual in not utilising Glc3Man9GlcNAc2-PPdolichol as the OST LLO donor substrate, but the selective utilisation of Man5GlcNAc2-PPdolichol and Man9GlcNAc2-PP-dolichol by TbSTT3A and TbSTT3B, respectively, means that this organism has evolved two simultaneously acting N-glycosylation pathways controlled by the amino acid environment of the N-glycosylation sequon (Fig 1).
Since T. brucei lacks an ER -mannosidase II, the N-glycosylation sites occupied by triantennary Man9GlcNAc2 transferred via TbSTT3B can only be processed as far as triantennary Man5GlcNAc2 by ER -mannosidase I.In other words, sites modified by TbSTT3B can only be occupied by triantennary Man9-5GlcNAc2 oligomannose glycans.On the other hand, sites occupied by biantennary Man5GlcNAc2 transferred via TbSTT3A can be processed to the paucimannose structure Man3GlcNAc2 by ER -mannosidase I and, from there, to a variety of complex N-glycan structures up to and including large multi-antennary pNAL (Gal1-4GlcNAc)n containing complex N-glycans with over 50 pNAL repeats [2] (Fig 1).
Why BSF trypanosomes have evolved to produce such unusually large and complex N-glycans is unclear.One proposal is that complex N-glycans containing pNAL repeats act as ligands for unspecified lectins that regulate in some way receptor-mediated endocytosis of scavenging receptors, such as the transferrin receptor (TfR) [18].The process of scavenging iron from host transferrin (Tf) is essential to maintain cellular homeostasis and is achieved in BSF cells by expression of a cell surface TfR to scavenge holo-transferrin from the host as an iron source [19].TfR has structural similarity to the major surface coat component of the parasite, the variant surface glycoprotein (VSG), but is a heterodimer of two proteins encoded by expression site associated genes ESAG6 and ESAG7 [20].TfR is bound to the cell surface via a single GPI-anchor on ESAG6 [21,22] and localises to the flagellar pocket (FP), an invagination of the plasma membrane located at the base of the flagellum.TfR is heavily N-glycosylated; ESAG6 is occupied by two Man4GlcNAc2 paucimannose and three Man5GlcNAc2 oligomannose N-linked glycans, whilst ESAG7 is modified by a single paucimannose and two oligomannose structures [22].Despite an earlier claim that TfR is devoid of pNAL containing N-glycans [22], more recent analysis demonstrated the reactivity of ESAG6 with lectins specific to pNAL modifications of complex N-glycans [23].Nevertheless, evidence for the necessity of linear pNAL modifications of TfR as sorting signals for lectin-mediated endocytosis is limited.This hypothesis is based primarily on the reduced uptake of transferrin (Tf) in cells pre-treated with chito-oligosaccharides (polymers of (-4GlcNAcβ1-)n) as surrogates of pNAL.Further, a counter argument for a key role for pNAL in the uptake of the essential nutrient Tf is that T. brucei BSF TbGT null mutants that affect the pNAL complex glycans [12,14,16,17] and BSF cells depleted of TbSTT3A by RNAi (lacking all or most complex N-glycans [5]) have no significant growth phenotypes in culture and the TbGT null mutants can all efficiently infect mice.Given this discrepancy, we considered that a more targeted mutational approach might resolve the issue.
To this end we generated a TbGT10 -/-/TbGT8 Flox/-conditional null mutant by diCre/LoxP mediated recombination in a TbGT10 -/-null mutant background [17].From this, we also isolated a TbGT10 -/-/TbGT8 -/-double null mutant.Based on previous structural characterisation of N-glycans from the TbGT8 -/-null [12,13] and TbGT10 -/-null [17] mutants, double deletion of TbGT8 and TbGT10 should, theoretically, prevent the synthesis of both linear pNAL chains and of 3,6 branch-points (Fig 4A).Here we describe the phenotype of BSF T. brucei simultaneously lacking TbGT8 and TbGT10 and discuss the implications for the role of pNAL synthesis in this organism.

Generation of a bloodstream form TbGT8 conditional null mutant in a TbGT10 null mutant background.
The generation of a TbGT10 null mutant (Δgt10::PAC/Δgt10 (SSU diCre)) in a cell line expressing diCre recombinase has been previously described [17] (Fig 2A).Starting with this parental cell line, the first TbGT8 allele was replaced by homologous recombination using a blasticidin deaminase (BSDr) resistance cassette flanked by tubulin regulatory elements to generate a TbGT8 +/-heterozygote.The remaining allele was replaced with a construct containing a TbGT8 gene flanked by loxP sites (floxed) in tandem with a hygromycin phosphotransferase-thymidylate kinase (HYG-TK) cassette for negative and positive selection, respectively [24].A Δgt10::PAC/Δgt10/Δgt8::BSDr/Δgt8::TbGT8-HYG-TK Flox [SSU diCre] clone was selected (hereafter referred to as TbGT10 -/-/TbGT8 Flox/-) expressing a single floxed copy of TbGT8.This cell line is a TbGT8 conditional null mutant in a TbGT10 null background, where TbGT8 can be excised upon addition of rapamycin.This was demonstrated by genomic DNA extraction and PCR analysis (Fig 2B).The data show the TbGT8 locus in wild type cells, the TbGT10 KO -/-/TbGT8 +/-mutant and two independent TbGT10 -/-/TbGT8 Flox/-conditional null mutant clones grown in the absence and presence of rapamycin for three days.The rapamycin-induced excision of the TbGT8 Flox allele in both clones is apparent (Fig 2B, 1.1 and 1.2).We also assessed the expected loss of hygromycin (HYG) resistance following rapamycin treatment.The majority of rapamycin-treated cells were killed by hygromycin, confirming high gene excision efficiency (Fig 2C).The TbGT10 -/-/TbGT8 Flox/-conditional null mutant showed similar growth kinetics in the absence and presence of rapamycin (Fig 2D).This indicates that, under in vitro culture conditions, BSF T. brucei can survive without TbGT8 and TbGT10 individually [12,17] and collectively (Fig 2D ).

A stable TbGT10 and TbGT8 double null mutant is viable in mice
A stable TbGT10 and TbGT8 double null mutant cell line was generated by treating TbGT10 -/- /TbGT8 Flox/-clone 1.1 cells with 100 nM rapamycin in the absence of hygromycin for 3 days and selecting clones by limiting dilution.The absence of both TbGT10 and TbGT8 was confirmed by hygromycin sensitivity and PCR amplification of their respective loci (Fig 3A ), alongside a control TbGT2b amplicon.A TbGT10 -/-/TbGT8 -/-double null mutant clone was selected for further analysis.The absence of both TbGT10 and TbGT8 was confirmed in this mutant by whole genome sequencing analysis (S1 Fig) .We analysed the infectivity of the TbGT10 -/-/TbGT8 -/-double null mutant in mice.No difference in the ability of wild type or TbGT10 -/-/TbGT8 -/-double null mutant cells to infect Balb/c mice was observed (Fig 3B).These data demonstrate that neither the individual [12,17] nor collective activities of TbGT8 and TbGT10 (Fig 3B) are essential for the survival of BSF T. brucei in vivo.
We performed lectin blotting with ricin agglutinin (RCA) on SDS cell lysates of wild type cells and of two independent TbGT10 -/-/TbGT8 Flox/-conditional null mutant clones grown without or with rapamycin for 3 days.We have previously shown that deletion of TbGT10 results in a reduction in RCA binding (implying a decrease in terminal βGal residues) and an alteration in the banding pattern of glycoproteins above ~70 kDa compared to wild-type [17].We observed the same alteration in RCA binding pattern in the lysates of both TbGT10 -/-/TbGT8 Flox/- conditional null mutant clones grown under permissive (-rapamycin) conditions compared to wild type (Fig 4B, compare lanes 2 and 4 with lane 1).The unaffected band at ~55 kDa band is most likely VSG221, which carries a terminal βGal residue on its GPI anchor [26].The most striking additional feature upon rapamycin-induced excision of TbGT8 in the TbGT10 null background was the appearance of a new RCA-binding band at ~60 kDa indicated by an asterisk (Fig 4B , lanes 3 and 5).The same effect was seen when comparing lysates from wild type, a TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions and the TbGT10 -/-/TbGT8 -/-double null mutant cells (Fig 4C).
Bloodstream form parasites express surface glycoproteins such as the ESAG6/7 heterodimeric transferrin receptor (TfR) and ISG65 that resolve by SDS-PAGE in the region of the RCA-binding ~60 kDa glycoprotein(s) from TbGT10 -/-/TbGT8 -/-cells.We therefore checked the status of these glycoproteins in lysates of wild type, a TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions and the TbGT10 -/-/TbGT8 -/-double null mutant cells (Fig 4C) by anti-ISG65 and anti-TfR Western blotting (S2 Fig) .This revealed that, in all cases, ISG65 runs at a distinctly higher apparent molecular weight than the RCA-binding ~60 kDa glycoprotein(s) from TbGT10 -/-/TbGT8 -/-cells.Further, it showed that ISG65 is not perturbed by the absence of TbGT10 or the absence of TbGT10 and TbGT8.This suggests that ISG65 lacks elaborated complex N-glycans.On the other hand, the anti-TfR Western blot showed that the absence of TbGT10 reduces the apparent molecular weight, and heterogeneity, of the ESAG6 subunit and that this is even more marked in the TbGT10 -/-/TbGT8 -/-double null cell line.The effects on TfR of TbGT10 were not explored in our previous TbGT10 study [17].However, in all cases, the TfR subunits did not comigrate with the RCA-binding ~60 kDa glycoprotein(s) from TbGT10 -/-/TbGT8 -/-cells.
To try to identify the RCA-binding ~60 kDa glycoprotein(s) from TbGT10 -/-/TbGT8 -/-cells, we performed hypotonic lysis of wild type and TbGT10 -/-/TbGT8 -/-cells to separate sVSG-enriched soluble fractions and cell ghost pellets.Proteins from each fraction were resolved by SDS-PAGE and subjected to RCA lectin blotting.The RCA-binding ~60 kDa glycoproteins enriched in the TbGT10 -/-/TbGT8 -/-cells were found predominantly in the supernatant fraction (S3A Fig) , suggesting that they are either GPI-anchored, and thus released in a soluble form upon hypotonic cell lysis (like sVSG), and/or lumenal soluble glycoproteins in the secretory or endosomal membrane system and released upon hypotonic organelle rupture.To identify them, we performed RCA-agarose pulldowns from the supernatant fractions of wild type, a TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions and TbGT10 - /-/TbGT8 -/-double null mutant cells.A preclearance step using anti-VSG IgG conjugated to Protein G agarose was performed to remove the majority of sVSG.The remaining RCA-binding glycoproteins were resolved by SDS-PAGE in triplicate for Coomassie staining and RCA lectin blotting with and without inhibitory sugars.The RCA reactive products specifically enriched in the TbGT10 -/-/TbGT8 -/-sample were excised from the gel, along with the equivalent sections from the wild type and TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions (S3B Fig) and processed for proteomic analysis by mass spectrometry.Protein groups enriched in the TbGT10 -/-/TbGT8 -/-sample, relative to the wild type and TbGT10 -/- /TbGT8 Flox/-conditional null mutant grown under permissive conditions samples, are indicated in (Table 1).Among these, two (ESAG2 and CBP1B) had predicted N-terminal signal peptides and 6 and 7 predicted N-glycosylation sites, respectively [27] (S4 Fig) .Both are known to resolve at high molecular weight by SDS-PAGE in WT BSF cell lysates [13,18].
From these data, we conclude that at least some glycoproteins are affected as predicted in (Fig 4A), collapsing from very high apparent MW to, in the case of ESAG2 and CBP1B, around 60 kDa apparent MW upon loss of both TbGT10 and TbGT8.Consistent with their appearance in the soluble fraction upon hypotonic lysis, ESAG2 is predicted to be a GPI-anchored protein [27] while CBP1B is a soluble lysosomal/endosmal serine protease [28].

The roles of TbGT10 and TbGT8 in poly-N-acetyllactosamine (pNAL) expression.
A characteristic of BSF T. brucei is the presence of very large pNAL containing N-linked glycans on glycoproteins of the flagellar pocket and endosomal and lysosomal compartments [1,2].To address the impact of removing TbGT10 and TbGT8 on pNAL expression, we performed tomato lectin (TL) blots of whole cell lysates, TL flow cytometry and methylation linkage analysis on glycopeptide preparations.
Blotting of whole cell lysates with biotinylated TL showed that deletion of TbGT10 (using the TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions) resulted in a significant reduction in the overall TL signal intensity for glycoproteins larger than 50 kDa Next, we quantitatively assessed the endocytic capacity of these parasites using flow cytometry to measure binding and uptake of fluorescent-conjugated TL as a surrogate marker for receptor-mediated endocytosis (Fig 5B and C).Cells were incubated with DyLight 488tomato lectin (TL::488) at 4°C to assess binding at the flagellar pocket without internalisation [29].The cells were subsequently transferred to either 14°C or 37°C for 10 minutes to activate endocytosis, directing TL::488 either to intermediate endocytic compartments or the terminal lysosome, respectively [29].At 4°C, TL binding remained unchanged in TbGT10 -/-/TbGT8 Flox/- conditional null mutant cells grown under permissive conditions but was reduced by 42% in TbGT10 -/-/TbGT8 -/-cells (p < 0.01), compared to wild type cells (Fig 5B and C).At 14°C and 37°C, TL uptake was significantly impaired in both TbGT mutants relative to wild type cells.In TbGT10 -/-/TbGT8 Flox/-conditional null mutant cells grown under permissive conditions, the reductions were 52% at 14°C, p = 0.0029 and 64%, p = 0.0078 at 37°C, while in TbGT10 -/- /TbGT8 -/-cells, the reductions were 64%, p = 0.0033 at 14°C and 79%, p = 0.0046 at 37°C (Fig 5C).The significant decrease in TL binding in the TbGT10 -/-/TbGT8 -/-cells at all temperatures, compared to TbGT10 -/-/TbGT8 Flox/-conditional null mutant cells grown under permissive conditions and wild type cells, suggests that TbGT8 has a more profound effect on TL-reactive pNAL expression in a cellular context than is discernible by lectin blotting.
The TL dependent parameters measured above are potentially complicated by the specificity of tomato lectin, which has high-affinity for linear pNAL structures of 3 or more [-3Galβ1-4GlcNAcβ1-] repeats [30] but which also binds to the N-acetyl-chitobiose core of oligomannose of N-linked glycans in lectin blots [31].To address this, we investigated the Nglycan structures synthesised by the TbGT10 -/-/TbGT8 -/-double null mutant compared to wild type using a chemical method.Following hypotonic lysis, to remove the majority of VSG as sVSG, washed cell ghost N-glycopeptide fractions were prepared by Pronase digestion, as previously described in [17] and subjected to methylation linkage analysis by gas chromatography-mass spectrometry (GC-MS).The partially permethylated alditol acetate (PMAA) derivatives were normalised to the PMAA corresponding to non-reducing-terminal mannose (t-Man) arising from oligomannose structures (Fig 6).As expected, the relative 3,6disubstituted Man (3,6-Man) derivative levels are similar, since 3,6-Man is present in both oligomannose and complex N-glycan structures.In contrast, there is a significant reduction in the relative levels of the Gal and GlcNAc PMAA derivatives, consistent with a reduction in Nglycan processing to large complex structures.Specifically, there is a reduction in t-Gal, 3-Gal, 6-Gal, 3,6-Gal and 4-GlcNAc residues consistent with a reduction in the UDP-GlcNAc : βGal β1-6 and β1-3 transferase activities of TbGT10 and TbGT8, respectively.However, the residual levels of 3-Gal, 6-Gal, 3,6-Gal residues make it clear that other TbGTs, most likely encoded within the CAZy GT67 family [1], can substitute to some degree for TbGT10 and TbGT8 and that the TbGT10 -/-/TbGT8 -/-double null mutant is, therefore, not devoid of pNAL containing Nlinked glycans (S6 Fig) .pNAL expression on the BSF transferrin receptor is greatly reduced in the TbGT10 -/-and TbGT10 -/-/TbGT8 -/-null mutants but transferrin-binding, localisation and receptor-mediated endocytosis are not affected.
The identification of CBP1B and ESAG2 in the RCA-binding 60 kDa apparent molecular weight material found in the TbGT10 -/-/TbGT8 -/-double null mutant (Fig 4B and C) suggested that some glycoproteins might be more affected than others with respect to pNAL content in the absence of TbGT10 and TbGT8.Further, a proposed role for pNAL modified N-glycans of BSF parasites is as sorting signal for lectin-mediated endocytosis of the TfR [18].We therefore compared the glycosylation status, TfR binding, localisation and receptor mediated endocytic properties of TfR in wild type, TbGT10 -/-/TbGT8 Flox/-conditional null mutant grown under permissive conditions and TbGT10 -/-/TbGT8 -/-double null mutant cells.
While newly synthesized ESAG6 and ESAG7 show weak TL reactivity via their oligomannose and paucimannose N-glycans, unlike ESAG7, only N-glycans of ESAG6 are extensively elaborated by addition of pNAL in the Golgi, enhancing their TL reactivity [23].Analysis of ESAG6 expressed in TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/-mutant cells by Western blotting suggested that maturation of ESAG6 by modification of its N-glycans with pNAL is impaired (S2 Fig) .To investigate this further, we treated affinity-purified TfR with endo-β-Nacetylglycosidase H (Endo H) to remove oligomannose N-glycans from wild type, TbGT10 -/- /TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/-mutant TfR preparations.After treatment, the samples were subjected to SDS-PAGE in duplicate and analysed by TL blotting without ( These results are fully in agreement with published work confirming that ESAG6 (which is strongly TL-reactive), unlike ESAG7 (which is weakly reactive), is modified by complex Nglycans containing linear pNAL [23].In contrast, the absence of TbGT10 in the TbGT10 -/- /TbGT8 Flox/-mutant or both TbGT10 and TbGT8 in the TbGT10 -/-/TbGT8 -/-mutant resulted in were also present in wild type samples.Importantly, the mobility of the non-specific bands did not overlap with the migration of ESAG6 in anti-TfR blots.Following TL blotting, nonspecific detection of recombinant Endo H was also observed and is indicated by asterisks.
Collectively, these data indicate that the TfR ESAG6 subunit made in the absence of TbGT10 alone or of TbGT10 and TbGT8 together does not undergo extensive glyco-processing to yield TL-binding, pNAL-containing N-glycans.Moreover, in the case of ESAG6, no compensatory TbGTs appear to be able to restore any processing of ESAG6 to carry TL-binding pNALcontaining N-glycans.
The fact that ESAG6 from the TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/-mutants failed to bind TL prompted us to investigate whether the absence of pNAL on TfR affects binding and uptake of its ligand.Lysates from wild and mutant cells were subjected to pulldowns with either anti-TfR antibodies or holo-Tf-coated beads. .The TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/- mutant cells showed approximately 30% and 42% reductions in Tf uptake at 14°C and 37°C relative to wild type, respectively.However, these differences were not statistically significant.
Given the variability of these experiments when cells were fixed prior to flow cytometry, we measured the actual rate of Tf:488 accumulation in live cells over 50 min and found no statistical difference in uptake between wild type and the TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/- /TbGT8 -/-mutant cells (Fig 8C).
Our flow cytometry assay is not sensitive enough to measure Tf binding at 4 o C [32], so we also assessed the number of binding sites using FITC:Tf.Here, we used wild cells and TbGT10 -/-, TbGT11 -/-and TbGT15 -/-null mutant cells generated in previous studies [14,16].TbGT11 and TbGT15 encode TbGnTI and TbGnTII, respectively, which add β1-2 linked GlcNAc residues to each arm of the Man3GlcNAc2 core, thus initiating complex N-glycan synthesis.Cells were incubated in the presence of FITC-conjugates, washed and cell extracts digested with Pronase to liberate FITC in order to measure total fluorescence [33] (Fig 8D).No statistically significant difference was detected in the binding of Tf between wild type and the three TbGT null mutants, indicating that altered pNAL synthesis in these cells has no detectable effect on cell surface Tf binding sites.
As an additional check for TfR function, the expression of RNA binding protein 5 (RBP5) was assessed in wild type cells and the TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/-mutants (Fig S7).RBP5 expression is upregulated upon iron starvation conditions, yet RBP5 levels were unchanged relative to wild type in our mutants, indicating that TfR endocytosis and iron scavenging is not impaired by pNAL loss [20,34,35].
Finally, to ensure that changes in the glyco-modifications of TfR did affect its cellular distribution, we performed immunofluorescent staining with anti-TfR antibodies.Microscopy revealed discrete foci between the central nucleus and the posterior kinetoplast, including a distinct signal in the flagellar pocket (Fig 8D , arrowheads) [32].This localisation is typical for functional TfR and appeared qualitatively similar in both mutants and WT, although there was a slight reduction in signal intensity in the TbGT mutants.Despite the subtle differences, overall, these results indicate that TfR function is not impaired following loss of the TbGTs.

High concentrations of N-acetylchito-oligosaccharides inhibit Tf receptor-mediated endocytosis
The hypothesis that TfR mediated endocytosis is pNAL-dependent is based principally on the reduced uptake of transferrin (Tf) in cells pre-treated with 15 mM of tri-N-acetyl-chitotriose and tetra-N-acetylchitotetraose [18].This hypothesis contradicts our findings that reduced pNAL on TfR does not significantly affect endocytosis of Tf.
To assess this, we sought to reproduce the finding in [18] and to investigate concentration dependence.We performed our standard endocytosis assay to quantify the uptake of TL and Tf (receptor-mediated endocytic cargo) and dextran (fluid-phase cargo) in wild type cells using three dilutions of chitin hydrolysate (a mixture of N-acetylchito-oligosaccharides; (-4GlcNAcb1-)n) (Fig 9).We found that uptake of both TL and Tf (Fig 9A and B), but not dextran (Fig 9C ), was inhibited at a 1:10 dilution of chitin hydrolysate.However, only TL uptake was inhibited at 1:100 and 1:1000 dilutions.These data reproduce the result in (18) that high concentrations of N-acetylchito-oligosaccharides inhibit Tf uptake but also show that lower concentrations of N-acetylchito-oligosaccharides do not, even when sufficient to block the binding of TL to cell surface (flagellar pocket) pNAL containing molecules.
Despite our inability to abolish pNAL synthesis entirely in the TbGT10 -/-/TbGT8 -/-double null cells, lectin blots for terminal β-Gal residues using RCA revealed that some glycoproteins were very substantially reduced in apparent molecular weight in the mutant.This included the essential cell surface-associated GPI anchored glycoprotein ESAG2 [36], known to normally bind to tomato lectin [18], and serine carboxypeptidase III (CBP1B) with likely lysosomal localisation based on analysis of a CPB1 homologue in T. cruzi [37].Both ESAG2 and CBP1B are predicted to be heavily N-glycosylated (Table 1

, S4 Fig) and have apparent molecular weights
of > 100 kDa in wild type cells [13,18], despite theoretical molecular weights of 56 and 52 kDa, respectively.Therefore, their detection at ~60 kDa in the TbGT10 -/-/TbGT8 -/-double null mutant is a likely consequence of the loss of all or most of their N-glycan pNAL modifications.
A similar phenomenon was also observed for the endosomal/lysosomal protein p67 on BSF cells, where the molecular weight of the mature glycoprotein was reduced by 50 kDa in the TbGT10 -/-null mutant [17,38].Further, the data presented in this paper show that the ESAG6 subunit of the BSF TfR is significantly reduced in apparent molecular weight and no longer binds TL in the TbGT10 -/-/TbGT8 Flox/-and TbGT10 -/-/TbGT8 -/-mutants.A similar conclusion with respect to ESAG6 glycosylation and ability of TfR was also reported in [39].Thus, all the evidence suggests that these proteins and others which are normally modified by polydisperse pNAL-containing complex N-glycans are still functional in the absence or depletion of pNAL structures.
The survival of the TbGT10 -/-/TbGT8 -/-double null mutant in vitro and in vivo, despite reductions in pNAL synthesis, appears contrary to the hypothesis of Nolan et al [18] which posits that receptor-mediated endocytosis of essential host molecules, like transferrin to supply iron, is pNAL-dependent.Instead, the main effect of deletion of TbGT8 and/or TbGT10 is the significantly impaired uptake of TL relative to WT but this is simply due to the reduction in TL binding sites.
The aforementioned pNAL-dependent endocytosis hypothesis was principally supported by the ability of 15mM N-acetylchitotriose and N-acetylchitotetraose to reduce the uptake of Tf by about 80%, and the uptake of low and high density lipoproteins (LDL and HDL) by about 50% [18].We were able to reproduce the effects on Tf uptake using high concentrations of chitin hydrolysate (1:10 dilution), which impaired both TL and Tf uptake into cells.By contrast fluid phase uptake, measured with dextran, was unaffected at the highest concentration of chitin hydrolysate (1:10).These data recapitulate a specific effect on TfR mediated Tf endocytosis at high N-acetylchito-oligosaccharide concentrations.However, at lower chitin hydrolysate concentrations (1:100 and 1:1000 dilution) TL uptake was still robustly inhibited, but not that of Tf.This shows that under conditions where endogenous TL-like lectins (like exogenous TL) are likely to be fully ligated by chitin-oligomers, there is little or no effect on Tf uptake.Taken together with the evidence presented here for pNAL-independence of Tf binding an uptake by the parasite TfR, we recommend that other explanations for the effects of high-concentrations of chito-oligosaccharides on Tf uptake should be entertained and caution exercised in making pNAL-dependent endocytosis a default hypothesis.
Finally, although the deletion of TbGT8 and/or TbGT10 has provided useful insights into trypanosome cell biology, and questioned prior assumptions, the precise role(s) of the giant, complex pNAL-containing N-glycan modifications in T. brucei remain obscure.

Cultivation of Trypanosomes-Trypanosoma brucei brucei
Lister strain 427 bloodstream form parasites, expressing VSG variant 221 (MiTat1.2) and transformed to stably express T7 polymerase and the tetracycline repressor protein under G418 antibiotic selection, were used in this study.This genetic background will be referred to from hereon as wild-type (WT).Cells were cultivated in HMI-11 medium containing 2.5 μg/mL G418 at 37 °C in a 5% CO2 incubator as described in [40].
DNA Isolation and Manipulation-Plasmid DNA was purified from Escherichia coli DH5α competent cells (New England Biolabs) using a Qiagen Miniprep kit.Gel extraction and reaction clean-up was performed using Qiaquick kits (Qiagen).Custom oligonucleotides were obtained from Thermo Fisher.T. brucei genomic DNA was isolated from ∼ 2 × 10 7 bloodstream form cells using a DNeasy Blood & Tissue Kit (Qiagen) using standard methods.

Generation of Gene Replacement Constructs-A full list and descriptions of all primers (S1
Table ) used in this study are available.The blasticidin deaminase (BSD) drug resistance cassette flanked by short regulatory elements from actin was generated by PCR-amplification using using Q5 DNA polymerase, a pNAT vector template and primers SMD402/3.~500 bp 5' and 3' flanking regions of Tb427.10.12290 (NEB) with primers SMD400/1 and SMD404/5 respectively using (Lister strain 427, variant 221 strain) genomic DNA as a template.Primers were designed with 20 nucleotide overlap regions to enable Gibson Assembly into a linear, pUC19 vector amplified using primers SMD33/4.Amplicons were PCR purified (Qiagen) and 10 pmol of each used in a 4-fragment Gibson assembly reaction (NEB) to generate TbGT8 5'_BSDr_TbGT8 3'_pUC19.The first allele of TbGT8 was replaced with the BSD drug resistance construct to generate a Δtbgt8::BSD/TbGT8 single deletion mutant.To generate a loxP-flanked TbGT8 expression cassette Q5 polymerase PCR amplification of Tb427.10.12290 containing a 3' stop codon using oligonucleotides SMD355/6 to confer 5' FseI and 3' BglIII cloning sites for cloning into the loxP vector to generate pSY45_pDS66 _TbGT8 Flox .This construct was used as a template for PCR amplification using SMD351/2 whilst 5'-637 bp and 3' 588 bp homologous flanks for Tb427.10.12290 were PCR amplified using SMD and SMD349/50 and SMD353/4, respectively.Each PCR amplicon contained 20 bp overlapping ends to facilitate Gibson assembly and generate TbGT8 5'_pSY45_pDS66_TbGT8 Flox _TbGT8 3'_pUC19.This construct was used to generate the Δtbgt10::PAC/Δgt10/Δtbgt8::BSD/TbGT8 Flox (SSU diCre) cell line hereon referred to as 'TbGT8 Flox ' conditional knockout mutants.
Transfection of bloodstream form T. brucei-Constructs for gene replacement and ectopic expression were purified, digested with appropriate restriction enzymes to linearize, precipitated, washed with 70% ethanol, and re-dissolved in sterile water.The linearized DNA was electroporated into T. brucei bloodstream form cells (Lister strain 427, variant 221) that were stably express T7 RNA polymerase and the tetracycline repressor protein under G418 selection.Cell transfection was carried out as described previously [40][41][42].
Induction of diCre mediated gene deletion-Mid-log stage TbGT10 -/-/TbGT8 Flox/-conditional null mutant cultures (~1 x 10 6 cells/mL) were passaged to 2 x 10 3 cells/mL or 2 x 10 4 cells/mL and dosed with 100 nM rapamycin (Abcam) from a 1 mM stock solution in DMSO.Cells at late-log phase were harvested for analysis after 3 or 2 days respectively.Conditional gene deletion was assessed by PCR amplification of genomic DNA using Taq polymerase (NEB) and oligonucleotides SMD357/8 flanking the TbGT8 locus.Hygromycin drug sensitivity was used as a proxy of TbGT8 Flox loss by seeding cells at a density of 2x 10 3 cells/mL and culturing for 3 days in the presence or absence of 100 nM rapamycin and/or hygromycin.Daily cell counting was performed to assess growth rates.A single clone (1.1) that gave a robust growth defect in the presence of hygromcyin was selected as the TbGT10 -/-/TbGT8 Flox/-conditional null mutant.
Generation of double null mutants-TbGT10 -/-/TbGT8 Flox/-conditional null mutant cells grown in the presence of 100 nM rapamycin for 3 days were serially diluted in 96 well plates in the presence of 100 nM rapamycin.Four clones were seeded at 2 x 10 3 cells/mL +/-hygromycin to confirm loss of TbGT8_TK-HYG Flox array by drug sensitivity.TbGT8 Flox loss was confirmed by PCR analysis and a single clone (1.1) lacking both TbGT10 and TbGT8 Flox (TbGT10 -/-/TbGT8 -/- double null mutant) was selected for further analysis.
Mouse infectivity studies-Animal studies were carried out under UK Home Office regulations (Project licence P4525BB4C) and the study plan approved by a Home Office Animals (Scientific Procedures) Inspector.3 groups of 5 female Balb/c mice each weighing between 18 -25g were housed in standard holding cages with water and food available ad libitum throughout the study.Wild-type and TbGT10 -/-/TbGT8 -/-double null mutant bloodstream form trypanosomes were grown in HMI-11T media, washed in media without antibiotics and re-suspended at 1 x 10 6 cells/mL.0.2 mL of the parasite suspension was injected intraperitoneally per animal.The ability of TbGT10 -/-/TbGT8 -/-double null mutant cells to establish infection in the blood relative to the WT control was assessed 1, 2 and 3 days post-infection by tail bleeding and cell counting using a Neubauer chamber in a phase contrast microscope.
Western/ Lectin blotting-For Western and lectin blot analysis, 5 x 10 6 -1 x 10 7 cells were lysed in lysis buffer (25mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100) and solubilised in 1xSDS sample buffer containing 0.1 M DTT by heating at 55°C for 20 min.Alternatively, cells were lysed by osmotic shock by incubating with water containing 0.1 mM TLCK, 1 µg/mL leupeptin and 1 µg/mL aprotinin (pre-warmed to 37°C) at 37 °C for 5 min.This releases all the cytosolic components and majority of VSG protein as soluble form VSG (sVSG).sVSG was liberated from cell ghosts by centrifugation at 12,000 g for 5 minutes and proteins extracted from the cell ghost pellet via lysis buffer and sample buffer extraction.Glycoproteins were resolved by SDS-PAGE (approx.1×10 7 cell equivalents/lane) on NuPAGE bis-Tris 4-12% gradient acrylamide gels (Invitrogen) and transferred either to nitrocellulose or PVDF membranes (Invitrogen).Ponceau S staining confirmed equal loading and transfer.
For TL blotting, glycoproteins were probed for 1 hour using biotinylated-TL (1:5000 dilution, Vector Laboratories Inc.) in 3% BSA blocking buffer.To confirm the specificity of TL binding, biotinylated-TL was pre-incubated with a 1:10 dilution of chitin hydrolysate (Vector Laboratories Inc.).Membranes were washed three times with 1X PBST and then incubated with anti-biotin at a 1:5000 dilution for 1 hour at room temperature.Ponceau S staining served as loading control.Blots in Figures 5, 6, and 7 were developed using Super Signal ECL reagent (Thermo Fisher Scientific) and imaged with a ChemiDoc Gel Imaging MP System (Bio-Rad).
Immunoblot analysis was carried out using Image Lab Software (Bio-Rad).Statistical analyses were performed by t-test in PRISM v10 (GraphPad Software, Inc., San Diego, CA).Differences were considered statistically significant at a p-value of <0.05.
Analysis of FITC-Tf binding-Protocol from [33].BSF T. brucei WT, TbGT10 KO, TbGT11 KO and TbGT15 KO cells were subbed at 1x10 5 cells /mL in 2 x 100 mL HMI-11 (10% FCS, no antibiotics) cultures and grown for 40 h prior to harvest.On the day of harvest 150 mL of each cell line was centrifuged at 1,000 g 10 mins at 4 o C and pellets re-suspended in 10 mL of ice-cold 1 X trypanosome dilution buffer (TDB; 5 mM KCl, 80 mM NaCl, 1 mM MgSO4,20mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose, pH 7.4) in 15 mL falcon tubes.Cells were washed by centrifugation at 900 g for 10 mins at 4 o C and resuspension in a further 10 mL of ice-cold 1X TDB and centrifuged once more.All the supernatant was removed, and the pellet of cells resuspended in 1 mL ice cold TDB.Cells were diluted 1 in 100 by inoculating 10 μL of cell suspension in 990 uL 1x TDB and counted on a haemocytometer.Cells were adjusted to 2x10 8 cells/mL by addition of ice-cold 1 x TDB.A sample of this cell suspension was frozen for protein assays.Aliquots of 500 uL containing 10 8 cells were transferred to 500 uL volumes containing 100 μg of FITC-holotransferrin (bovine).After incubating on ice for 15 minutes, cells were centrifuged at 11,700 x g for 30 sec at 4 o C.After removal of the supernatants, pellets were resuspended in 1 mL ice-cold TDB and centrifuged again at 11,700 x g for 30 sec at 4 o C.After discarding the supernatants, cells were resuspended in 1 mL ice-cold TDB and centrifuged again at 11,700 x g for 30 sec at 4 o C. Supernatants were then discarded, and the cell pellets frozen for proteolysis.FITC-holotransferrin standards for proteolysis were prepared by diluting 20 μL of the FITC-protein stock solution with 180 uL TDB to give final concentrations of 188.8 µg/mL and 183.3 µg/mL, respectively.
After incubation, the cells were washed with cold 1x HBSG, fixed with 1% formaldehyde (SIGMA), and analysed by flow cytometry (10,000 cell counts/condition) using a BD LSRFortessa.Data were analysed using FlowJo (Tree Star).For uptake in live cells, 5 µg/ml TL TexasRed (Invitrogen) or 10 µg/ml hTf AF-647 (Jackson ImmunoResearch Laboratories, Inc.) was added and cells were incubated at 37 o C for 30 minutes.Flow cytometry analyses were identical to fixed cells.
Affinity purification of TfR and EndoH treatment-TfR was affinity-purified using either anti-TfR antibodies or holo-transferrin-coated beads, following the methodologies described in [22,23].A total of 1x10 7 cells were harvested and lysed in 1X RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5 % Na deoxycholate).1% SDS and 1 MM EDTA) containing the protease inhibitor cocktail described above.After clearing the lysates by centrifugation, the supernatant was mixed with either anti-TfR antibodies or a 50% slurry of Tf-beads and incubated overnight at 4°C.After washing 3X with 1X RIPA buffer, the bound TfR was digested on beads with EndoH according to the manufacturer's recommendations (New England Biolabs).Eluates were analysed by lectin or Western blotting as described above.

N-glycopeptide preparation for GC-MS analysis-To prepare N-glycopeptides of T. brucei
bloodstream-form wild-type and TbGT10 -/-/TbGT8 -/-double null mutant cells, 5 x 10 9 cells were harvested and washed twice with 5 mM KCl, 80 mM NaCl, 1 mM MgSO4, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose (pH 7.4) and depleted of sVSG using hypotonic lysis in 5 mL water (5 min, 37 o C).The sVSG depleted cell ghost pellets were recovered by centrifugation (12000 x g, 30 min, 4 o C) and resuspended in 1.5 mL of 20 mM ammonium bicarbonate and mixed with 50 µl of 10 mg/mL of freshly prepared Pronase (Calbiochem, #53702) dissolved in 5 mM calcium acetate and digested at 37 °C for 24 h.The digest was centrifuged at 12000 x g for 30 min to remove the cell ghost membranes and nuclei and the supernatant was incubated at 95 °C for 20 min to heat inactivate the Pronase and again centrifuged to remove particulates.The supernatant containing the Pronase digested glycopeptides was applied to a 30 kDa cut-off centrifugal filter (Amicon) and diafiltrated with water three times.The resulting aqueous filtrate was subjected to chloroform phase separation by mixing with equal volume of chloroform to remove any remaining lipid contaminants.The upper aqueous phase (Pronase glycopeptide fraction) was collected in a fresh tube and used for GC-MS methylation linkage analysis.
Methylation linkage analysis-Samples were dried and subjected to permethylation using the sodium hydroxide method as described in [43].The permethylated glycans were then subjected to acid hydrolysis, NaB[ 2 H]4 reduction, and acetylation to generate partially methylated alditol acetates (PMAAs) [43].The PMAAs were analysed by GC-MS (Agilent Technologies, 7890B Gas Chromatography system with 5977A MSD, equipped with Agilent HP-5ms GC Column, 30 m X 0.25 mm, 0.25 µm).   A. Gene replacement strategy using a previously described TbGT10 null cell line (TbGT10 - /-) [17] which constitutively expresses diCre recombinase from the ribosomal small subunit (SSU).This was used to generate a TbGT8 conditional knockout cell line lacking TbGT10 (referred to in the text as TbGT10 -/-/TbGT8 Flox/-conditional null).The first allele of TbGT8 was replaced using a blasticidin resistance gene (BSDr) to generate TbGT10 -/-/TbGT8 +/-mutants.To make a conditional null mutant of TbGT8 the transgene was introduced at the second allele using a dicistronic construct containing a hygromycin resistance (HYG) Thymidine kinase (TK) fusion flanked by loxP (black arrows) sites (TbGT8 Flox ).B. PCR was performed using gDNA harvested 72 h after rapamycin treatment (+ Rap) and oligonucleotide primers SMD357 and 358 (open arrows) that anneal outside of the homologous recombination site.Expected amplicon sizes are underlined.Resolution of PCR products by agarose gel electrophoresis confirms the replacement of endogenous TbGT8 by TbGT8 Flox and the excision of TbGT8 Flox upon rapamycin treatment.Two TbGT10 -/-/TbGT8 Flox/-clones (1.1 and 1.2) are shown.Wildtype (WT) and TbGT10 -/-/TbGT8 +/-mutants were included as controls.C. Growth of TbGT10 -/- /TbGT8 Flox/-conditional null mutant cells cultured with (+Rap) or without (−Rap) 100 nM rapamycin for 3 days.Cells were seeded in the presence or absence of hygromycin (HYG) to assess floxed gene loss by hygromycin sensitivity.Data are means ± SD (n=4 clones with 3 technical replicates per n) D. Growth kinetics of TbGT10 -/-/TbGT8 Flox/-conditional null mutant cells grown with or without (control) 100 nM rapamycin for 7 days.Cells were seeded at 2 × 10 3 cells/ml and diluted to 2 × 10 4 cells/ml every 2 days from day 3. Cell density was determined by counting at 24 h intervals.Data are means ± SD (n = 4 clones with 3 technical replicates each).

RCA pulldown and protein identification
lane 2) compared to wild type (Fig 5A, lane 1).The reduction in TL binding is incomplete, however, with chitin hydrolysate-inhibitable signals remaining at around 75, 100, 150 and >250 kDa apparent molecular weight (Fig 5A, lane 2).The further ablation of TbGT8 in this TbGT10 null background had little discernible effect on the TL blot pattern (Fig 5A, lane 3).The signals below 50 kDa correspond to endogenously biotinylated proteins, and are not inhibited by chitin hydrolysate.
Fig 7, top panel, 1 o TL blot) or with competing chitin hydrolysate (Fig 7, bottom panel, 1 o TL/chitin blot) to confirm TL binding specificity.All primary blots were re-blotted with anti-TfR (2 o TfR blots, top and bottom panels) to ensure efficient pulldowns and Endo H digestion.In wild type samples, both Endo H treated (partially deglycosylated ESAG6; dESAG) and untreated ESAG6 showed strong reactivity in primary TL blots with a banding pattern and mobility that overlapped with the corresponding ESAG6 signals in anti-TfR secondary blots (Fig 7, compare lanes 1 and 3, 2 and 4; ESAG6 vs dESAG6).Endo H untreated ESAG7 was weakly reactive, while Endo H treated showed no reactivity (Fig 7, compare lanes 1 and 3, 2 and 4; ESAG7 vs dESAG7).The Endo H treated partially deglycosylated ESAG7 (dESAG7) (Fig 7, lane 2) signal does not overlap with the corresponding dESAG7 anti-TfR signal (Endo H+, lane 4).
loss of TL reactivity in ESAG6 (Fig 7, lanes 5, 6, 9, 10).The 2 0 aTfR blots (Fig 7, lanes 3,4,7,8,11,12) act as TfR loading and Endo H digestion controls.We attribute certain bands observed in the TL blots to non-specific cross-reacting bands (Fig 7, arrowheads), as these The pulldowns were resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-TfR (Fig 8A).A number of conclusions can be made from these data: First, the anti-TfR blot revealed no obvious differences in the steady-state levels of the ESAG6 and ESAG7 TfR subunits (Fig 8A, compare lanes 1, 2, 3), suggesting that lack of pNAL does not affect TfR protein stability.Second, only a fraction of total TfR associates with Tf-coated beads, consistent with the notion that some TfR was already saturated with Tf from the media prior to the pulldowns (Fig 8A, compare lanes 1-3 vs 4-6) [32].Third, association to Tf beads confirmed the presence of functional heterodimers of ESAG6 and ESAG7 subunits, indicating that an absence of pNAL on N-glycans does not affect TfR interaction with Tf in vitro.Fourth, the decrease in the size of ESAG6 in the mutants relative to wild type (Fig 8A, compare lane 1 vs 2 and 3; lane 4 vs 5 and 6) confirm that removal of TbGT10 alone or TbGT10 and TbGT8 together result in limited glycoprocessing of ESAG6.Next, we used flow cytometry to quantify uptake of fluorescent holotransferrin (Tf::488) in a large number of single cells in vivo (Fig 8B) of RCA reactive product-Soluble VSG fraction (sVSG) from 5 x 10 7 cell equivalents were collected and incubated with anti-VSG221 conjugated to Protein G Agarose (Pierce) for 1 hours at room temperature with rotation to deplete the sVSG of VSG221.Supernatants were removed from VSG221-beads by centrifugation and incubated with RCA-coupled Agarose (Vector Labs) overnight at 4 o C with gentle rotation.The following day the RCA-agarose beads were washed three times in 1 x PBS and bound proteins eluted by heating in 1 x SDS sample buffer at 55 o C for 30 minutes.Proteins were resolved by SDS-PAGE and either fixed using Quick Coomassie Stain (Neo Biotech) (approx.2×10 7 cell equivalents/lane) or transferred to NTC membranes (approx.1.4×10 7 cell equivalents/lane) and subjected to RCA lectin blotting before or after incubation with 30 mg/mL D-galactose and 30 mg/mL to localise the RCA reactive band.Slices corresponding to molecular weight of the RCA reactive band were cut from the Coomassie stained gel for LC-MS/MS protein identification at the FingerPrints Proteomics Facility (College of Life Sciences, University of Dundee).

Figure 2 :
Figure 2: Generation of a bloodstream form TbGT8 conditional null mutant in a TbGT10 null mutant background.A. Gene replacement strategy using a previously described TbGT10 null cell line (TbGT10 - /-)[17] which constitutively expresses diCre recombinase from the ribosomal small subunit (SSU).This was used to generate a TbGT8 conditional knockout cell line lacking TbGT10 (referred to in the text as TbGT10 -/-/TbGT8 Flox/-conditional null).The first allele of TbGT8 was replaced using a blasticidin resistance gene (BSDr) to generate TbGT10 -/-/TbGT8 +/-mutants.To make a conditional null mutant of TbGT8 the transgene was introduced at the second allele using a dicistronic construct containing a hygromycin resistance (HYG) Thymidine kinase (TK) fusion flanked by loxP (black arrows) sites (TbGT8 Flox ).B. PCR was performed using gDNA harvested 72 h after rapamycin treatment (+ Rap) and oligonucleotide primers SMD357 and 358 (open arrows) that anneal outside of the homologous recombination site.Expected amplicon sizes are underlined.Resolution of PCR products by agarose gel electrophoresis confirms the replacement of endogenous TbGT8 by TbGT8 Flox and the excision of TbGT8 Flox upon rapamycin treatment.Two TbGT10 -/-/TbGT8 Flox/-clones (1.1 and 1.2) are shown.Wildtype (WT) and TbGT10 -/-/TbGT8 +/-mutants were included as controls.C. Growth of TbGT10 -/- /TbGT8 Flox/-conditional null mutant cells cultured with (+Rap) or without (−Rap) 100 nM rapamycin for 3 days.Cells were seeded in the presence or absence of hygromycin (HYG) to assess floxed gene loss by hygromycin sensitivity.Data are means ± SD (n=4 clones with 3 technical replicates per n) D. Growth kinetics of TbGT10 -/-/TbGT8 Flox/-conditional null mutant cells grown with or without (control) 100 nM rapamycin for 7 days.Cells were seeded at 2 × 10 3 cells/ml and diluted to 2 × 10 4 cells/ml every 2 days from day 3. Cell density was determined by counting at 24 h intervals.Data are means ± SD (n = 4 clones with 3 technical replicates each).

Figure 5 :
Figure 5: TbGT knockout mutants are deficient in TL binding and uptake.A. Whole cell lysates(10 7  cell equivalents per condition) from WT, TbGT10 -/-/TbGT8 Flox/-conditional null mutant and TbGT10 -/- /TbGT8 -/-double null mutant cell lines were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with TL::Biotin only (TL, top left) or with TL::Biotin pre-incubated with chitin hydrolysate at 1:10 dilution (TL/chitin, top right) as competitive inhibitor.Membranes were counter-stained with Ponceau S as loading control (bottom panels).B. Binding and internalisation of TL::Dylight488, acting as a surrogate for receptor-mediated endocytic cargoes, were measured by flow cytometry.Cells were incubated with TL::Dylight488 at 4 °C for 5 min and then transferred to 14 or 37 °C for 10 min to activate endocytosis.Histogram profiles show fluorescent intensities distributions of 10,000 WT, TbGT10 -/-/TbGT8 Flox/-conditional null mutant and TbGT10 -/-/TbGT8 -/-double null mutant cells.C. Bar chart shows median fluorescence intensities (MFI, arbitrary units) for each population of cells, presented as means ± SD, n = 3 biological replicates.Unpaired t-test with Welch's correction indicated significant differences (*P < 0.0339, **P < 0.008) between WT and each of the mutant cell lines at the specified temperatures.Chitin hydrolysate at 1:10, 1:100 and 1:000 dilution (inhibitor) was used as specificity control for each temperature and the MFI values were < 22 arbitrary units.

Figure 9 :
Figure9: Effect of chitin hydrolysate on TL, Tf and Dextran uptake.Live, wild-type bloodstream form cells were incubated with (A) Alexa647-conjugated tomato lectin (TL::647), (B) Alexa647-conjugated transferrin (Tf::647), or with (C) Alexa488::Dextran at 37°C for 10 min to allow uptake.This was done in the absence (-CH, unstained, grey) or presence of varying dilutions (1:10 to 1:1000, black) of Chitin hydrolysate (+) as indicated in the plots (insets).The actual concentration of CH used is unknown, as this information is proprietary to the manufacturer of the product (Vector Laboratories, 2BScientific, UK).Histogram profiles show fluorescent intensities distributions of 10,000 cells per condition analysed by flow cytometry.Inhibition of TL and Tf uptake by chitin hydrolysate at a 1:10 dilution was consistent across three biological replicates in Figures5 and 7, respectively.The Dextran experiment was performed once as control for fluid phase endocytosis.