Samuel Martin Duncan

The gene TbFUT1 encodes an essential fucosyltransferase which, unexpectedly, localises to the mitochondrion of the protist parasite Trypanosoma brucei . The expression of TbFUT1 is required for the maintenance of mitochondrial membrane potential (ΨΔm) in the bloodstream form (BSF) of the parasite, but the precise functions of TbFUT1 are unknown. Here, we demonstrate that depletion of TbFUT1 causes the accumulation of dyskinetoplastid cells; i.e., cells lacking concatenated complexes of mini- and maxicircle kinetoplast DNA (kDNA), the mitochondrial DNA of these organisms. Morphological analysis by serial face block-scanning electron microscopy showed that the dyskinetoplastid mitochondria were otherwise unperturbed with respect to structure and volume. Proteomics analyses showed that TbFUT1 depletion caused a decrease in the steady-state levels of several subunits of the F _o -subcomplex and peripheral stalk components of the mitochondrial F _o F ₁ -ATP synthase, as well as a pronounced reduction in mitochondrial ribosomal large subunit (LSU) proteins and more minor reduction in small subunit (SSU) proteins. TbFUT1 was rendered redundant with respect to cell survival and ΨΔm generation upon F ₁ -γ ^WT/L262P mutation; a mutation that allows the generation of ΨΔm in the absence of mitochondrial translation. Additionally, depletion of TbFUT1 no longer perturbs kDNA replication in these cells, indicating that dyskinetoplasty is a downstream consequence of impaired ΨΔm. Depletion of TbFUT1 in wild type cells leads to the collapse of ΨΔm via a functional F _o F ₁ -ATP synthase complex. We therefore conclude these mutants are inhibited in the synthesis of F _o -subcomplex components and, thus, impairing the assembly of functional F _o F ₁ -ATP synthase complexes. Curiously, mitochondrial transcript levels exhibit similar changes in abundance after FUT1 ablation in the parental and F ₁ -γ ^WT/L262P mutants. Further, the ∼5-fold overexpression of TbFUT1 in the TbFUT1 conditional knockout mutant under permissive conditions selectively inhibits the formation of the fully RNA-edited A6 transcript by an unknown mechanism, partially suppressing F _o F ₁ -ATP synthase assembly in these mutants. Together, these data suggest that mitochondrial fucosylation is essential for the assembly of protein complexes containing kDNA encoded subunits.

Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic β3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed.

Trypanosoma brucei has large carbohydrate extensions on its N -linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasite’s glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative β3GT gene, TbGT10 . The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N -glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (− 6 Galβ1-4GlcNAcβ1-) _{≥ 4} poly- N -acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6- O -substituted- and 3,6-di- O -substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : βGal β1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N -glycans and GPI sidechains, respectively. The β1-6 specificity of this β3GT gene product and its dual roles in N -glycan and GPI glycan elaboration are notable.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9–1–1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9–1–1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.

Breeding for true loose smut (Ustilago nuda (Jens.) Rostr.) resistance in barley is expensive because of the great requirements for time, labour, and growth space, and thus is an ideal candidate for screening by indirect methods. One gene (Un8) confers resistance to most known races of the true loose smut pathogen and is the gene present in the majority of western Canadian barley cultivars. Here we report on linkage between this gene and a restriction fragment length polymorphism marker, or various forms of the marker, in a number of segregating barley populations. The different forms of the marker were developed through sequencing and postamplification restriction. The resulting allele-specific amplicon markers can be used in a relatively simple assay, based on the polymerase chain reaction, specific for either the resistant or susceptible allele. The marker was linked to the resistance gene in five crosses involving two sources of resistance and was located on chromosome 5 (1HL). The allele-specific amplicons are being routinely used for marker-assisted selection in conjunction with a rapid DNA template preparation method.