Homoserine and quorum-sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect-stage Trypanosoma brucei

Abstract

De novo synthesis of threonine from aspartate occurs via the β-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the β-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.

Figure 1

De novo threonine biosynthesis pathway. Aspartate is sequentially converted to homoserine via a series of enzymatic reactions involving aspartokinase (AspK), aspartate semialdehyde dehydrogenase (AspSD) and homoserine dehydrogenase (HSD). Homoserine is phosphorylated by HSK to form O-phospho-homoserine, a substrate for threonine synthase (ThrS) to produce threonine. Candidate genes for each of these metabolic enzymes are shown for Trypanosoma brucei and Leishmania major.

Figure 2

Multiple sequence alignment of HSK from representative species using CLUSTALW. Species abbreviations and NCBI accession numbers: Mj, Methanococcus jannaschii (Q58504); At, Arabidopsis thaliana (AAD33097); Ec, Escherichia coli (YP_488309); Sc, Saccharomyces cerevisiae (NP_011890); Tb, Trypanosoma brucei (XP_845560). See text for details on highlighted residues.

Figure 3

Kinetic characterisation of TbHSK. A. Purity of SDS-PAGE of recombinant TbHSK peaks eluted from the nickel affinity column. B. Rate of the NADH oxidation over a range of TbHSK concentrations. C. pH optimum of TbHSK for L-homoserine determined using constant ionic-strength buffers. D. K_m^app of TbHSK for L-homoserine under quasi-physiological conditions.

Figure 4

Genotypic analysis of WT, SKO and DKO cell lines. Southern blot analysis of AatII-digested genomic DNA (∼5 μg) from diploid wild-type trypanosomes (WT), ΔHSK::HYG (SKO^HYG); ΔHSK::PAC (SKO^PAC); and ΔHSK::HYG/ΔHSK::PAC (DKO) using a probe against the 3′-UTR of HSK (panel A) or the open reading frame of HSK (panel B). A schematic representation of the HSK locus and its gene replacements is shown to the right of the blot.

Figure 5

Growth analyses of bloodstream WT and DKO cells in vitro. A. WT (circles) and DKO (squares) cells cultured in TBM^TD with (open symbols) or without (closed symbols) 800 μM threonine. No viable cells visible after 4 days (). B. Growth of WT (open circles) and DKO (closed circles) cells in varying concentrations of L-threonine.

Figure 6

Growth analyses of procyclic WT and DKO cells in vitro. A. The cumulative growth curves of WT (open circles) and DKO (closed circles) cells cultured in DTM^TD supplemented with 400-μM threonine were determined over 2 weeks. WT (open squares) and DKO (closed squares) cells perished on day 6 in the absence of L-threonine (). B. Growth of WT (open circles) and DKO (closed circles) cells in varying concentrations of threonine. C. Growth of WT (open circles) and DKO (closed circles) cells in varying concentrations of L-homoserine. D. Growth of WT cells in varying concentrations of L-homoserine lactone (open circles); N-3-oxododecanoyl-L-homoserine lactone (closed circles); N-3-oxodecanoyl-L-homoserine lactone (open squares); and N-3-oxohexanoyl-L-homoserine lactone (closed squares). DKO cells failed to grow with any of these supplements.

Figure 7

Growth of Leishmania donovani promastigotes cultured in medium lacking aspartate and threonine. Cumulative growth curves were determined over 14 days with subculture into fresh medium every 2 days. No additions (open circles); plus 100 μM L-aspartate (closed circles); plus 400 μM L-threonine (closed squares); plus 100 μM L-aspartate and 400 μM L-threonine (open squares).

Figure 8

Enzymes predicted by bioinformatic analysis to be present in Sodalis glossinidius, Wigglesworthia glossinidia and Trypanosoma brucei. Key end-products of metabolism are boxed and excreted metabolites boxed and shaded. See legend for further details.