Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

Abstract

The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDP-glucose 4'-epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4'-epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4'-epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis.

Figure 1

clustal v alignment of T. brucei (Tb), E. coli (Ec), Saccharomyces cerevisiae (Sc), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), and human (Hu) UDP-Glc-4-epimerase predicted amino acid sequences. The reverse-text indicates residues that match the consensus sequence.

Figure 2

Southern blot of T. brucei procyclic genomic DNA digested with a panel of restriction endonucleases and probed for T. brucei galE. Approximately five micrograms of DNA, digested overnight with BamHI, NcoI, PstI, SacI, and XhoI restriction enzymes, was loaded per lane. Hybridization was at 60°C overnight. The blot was washed with 1× SSC (1× SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7)/0.1% SDS for 20 min at 60°C, followed by 0.5× SSC/0.1% SDS for 20 min at 60°C. The single band in each lane shows that the epimerase is present as a single copy per haploid genome.

Figure 3

Functionality of the T. brucei galE gene and recombinant protein. (A) Functional complementation of a galE-deficient strain of E. coli. Cells expressing T. brucei galE (RF0) from pUC18 formed dark red colonies on McConkey agar. When the epimerase is not expressed and the cells grow poorly. (B) Purification of recombinant T. brucei UDP-Glc-4′-epimerase; 10% SDS/PAGE gel stained with Coomassie blue of total E. coli lysate (lane 1), soluble supernatant (lane 2), Ni²⁺-Sepharose purified protein (lane 3), and dialyzed purified protein (lane 4). (C) Lineweaver–Burk plot of purified recombinant T. brucei epimerase activity.

Figure 4

Construction of T. brucei conditional galE null mutants. (A) Schematic representation showing the location of the TETR gene in the “wild-type” cells, the targeted replacement of the one galE allele with PAC, the introduction of an ectopic tetracycline-inducible copy of galE into the rDNA locus, and replacement of the second galE allele with HYG. The primers indicated are those used to amplify regions of interest in (Fig. 5). (B) Southern blot of conditional galE null mutants and intermediate cell lines. Aliquots (≈5 μg) of PstI-digested DNA were blotted with the T. brucei galE probe (Left) or the 5′-UTR probe (Right). The DNA was from wild-type galE^+/+ cells (lane 1), ΔgalE∷HYG cells (lane 2), ΔgalE∷PAC cells (lane 3), galE^Ti ΔgalE∷HYG cells (lane 4), galE^Ti ΔgalE∷PAC cells (lane 5), and conditional galE^Ti ΔgalE∷PAC/ΔgalE∷HYG null mutant clones derived from galE^Ti ΔgalE∷HYG cells (lane 6) and from galE^Ti ΔgalE∷PAC cells (lanes 7 and 8). The probe reveals allelic (galE) copies at 4 kb and ectopic tetracycline-inducible (galE^Ti) copies at 8 kb. The 5′-UTR probe shows differences when the allelic galE copies are replaced by the drug resistance genes. Note that the HYG gene contains a PstI site and therefore shows a larger change.

Figure 5

Growth and Northern blots of the T. brucei conditional galE null mutants. Representative growth curves for one of the three conditional mutant clones are shown. Cells grown in the presence of tetracycline were washed at day 0 and reintroduced into tetracycline-containing medium (A; +Tet) or tetracycline-free medium (B and C; −Tet). Tetracycline was reintroduced into one of the cultures after 14 days (C). (D) Northern blot of total RNA prepared from “wild-type” T. brucei (lane 1) and conditional galE null mutants grown with (+Tet; lanes 2–5) and without (−Tet; lanes 6–9) tetracycline for 0, 8, 24, and 48 h and from cells that spontaneously re-emerged after 16 days without tetracycline (lane 10). The blot was probed with a T. brucei galE probe (Upper) and a β-tubulin probe (Lower).

Figure 6

The mechanism of the recovery of the T. brucei conditional galE null mutants in the absence of tetracycline. (A) Ethidium-bromide-stained agarose gel of PCR products of the TetOp and TETR regions amplified from genomic DNA from conditional mutant cells from three cultures that recovered after 16 days (lanes 1–3), conditional mutant cells induced to grow after the reintroduction of tetracycline after 14 days (lane 4), conditional mutant cells grown continuously in the presence of tetracycline (lane 5), “wild-type” (TETR⁺) cells grown without tetracycline (lane 6), and “wild-type” (TETR⁺) cells grown with tetracycline (lane 7). (B) Southern blot of genomic DNA digested with NotI and SalI from the same cells described in A with TETR and NEO probes.