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Erythritol Feeds the Pentose Phosphate Pathway Via Three New Isomerases Leading to D-Erythrose-4- Phosphate in Brucella

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Erythritol Feeds the Pentose Phosphate Pathway Via Three New Isomerases Leading to D-Erythrose-4- Phosphate in Brucella Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4- phosphate in Brucella Thibault Barbiera,1, François Collardb,1, Amaia Zúñiga-Ripac, Ignacio Moriyónc, Thibault Godardd, Judith Beckere, Christoph Wittmanne, Emile Van Schaftingenb,2,3, and Jean-Jacques Letessona,2,3 aResearch Unit in Microorganisms Biology, University of Namur, B-5000 Namur, Belgium; bWelbio and de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium; cDepartamento de Microbiología e Instituto de Salud Tropical, Universidad de Navarra, 31008 Pamplona, Spain; dInstitute of Biochemical Engineering, Technische Universität Braunschweig, 38106 Braunschweig, Germany; and eInstitute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany Edited by Hans Leo Kornberg, Boston University, Boston, MA, and approved November 3, 2014 (received for review July 31, 2014) Erythritol is an important nutrient for several α-2 Proteobacteria, injections of erythritol increase the numbers of Brucella in the spleens including N2-fixing plant endosymbionts and Brucella, a worldwide of intraperitoneally infected calves (3, 7). For these reasons, erythritol pathogen that finds this four-carbon polyol in genital tissues. has been considered a major factor in Brucella pathogenesis. Erythritol metabolism involves phosphorylation to L-erythritol- Erythritol metabolism (Fig. 1A, accepted pathway) starts with 4-phosphate by the kinase EryA and oxidation of the latter to its phosphorylation by EryA to yield L-erythritol-4-P (10, 11), L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted which is then oxidized by the dehydrogenase EryB to L-3-tetrulose- that further steps involve oxidation by the putative dehydrogenase 4-P (10). It is accepted that the next three steps imply the oxida- EryC and subsequent decarboxylation to yield triose-phosphates. Ac- tion of the first carbon of L-3-tetrulose-4-P to an aldehyde by an cordingly, growth on erythritol as the sole C source should require NAD-dependent dehydrogenase, purportedly EryC (12), followed aldolase and fructose-1,6-bisphosphatase to produce essential by dehydrogenation and decarboxylation reactions generating di- hexose-6-monophosphate. However, we observed that a mutant hydroxyacetone-P and carbon dioxide as end products (10). devoid of fructose-1,6-bisphosphatases grew normally on eryth- The use of this pathway to grow on erythritol is not restricted ritol and that EryC, which was assumed to be a dehydrogenase, to brucellae or to their intracellular pathogenic lifestyle but is actually belongs to the xylose isomerase superfamily. Moreover, we shared by other members of the Rhizobiales,suchasOchrobactrum found that TpiA2 and RpiB, distant homologs of triose phosphate anthropi (an environmental bacteria), Rhizobium leguminosarum, isomerase and ribose 5-phosphate isomerase B, were necessary, as and Sinorhizobium meliloti (both plant symbionts) (13, 14). In all previously shown for Rhizobium. By using purified recombinant these bacteria, there is, downstream of the ery operon, a second enzymes, we demonstrated that L-3-tetrulose-4-phosphate was highly conserved operon that contains three ORFs including converted to D-erythrose 4-phosphate through three previously tpiA2 and rpiB, which are homologous to triose phosphate isom- unknown isomerization reactions catalyzed by EryC (tetrulose-4- erase and ribose 5-phosphate isomerase B, respectively (13). In- phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; terestingly, rpiB and tpiA2 mutants of R. leguminosarum (15) are renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; re- named EryI), a pathway fully consistent with the isotopomer distribu- unable to grow on erythritol as a carbon source. tion of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. Significance D-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6- Erythritol is a preferential substrate for Brucella, a common zoo- phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the notic bacterial pathogen. This four-carbon polyol is found in the first description to our knowledge of a route feeding carbohydrate reproductive organs of several affected species, a feature that metabolism exclusively via D-erythrose 4-phosphate, a pathway that may account for the characteristic viscerotropism of Brucella that may provide clues to the preferential metabolism of erythritol by leads to sterility and abortion. Although described previously as Brucella and its role in pathogenicity. feeding glycolysis via dihydroxyacetone-phosphate, we show here that erythritol is actually converted into D-erythrose-4- Brucella | erythritol | alphaproteobacteria | pentose phosphate cycle | phosphate through a hitherto undescribed set of reactions that tetrose metabolism involves three isomerases and that allows hexose-mono- BIOCHEMISTRY phosphate synthesis and growth by feeding the pentose phos- rucellosis is a widespread bacterial zoonosis caused by the phate shunt. Elucidation of this unique carbohydrate pathway, Bfacultative intracellular pathogens Brucella spp. Domestic which also applies to the Rhizobiales plant endosymbionts, opens ruminants and swine, which altogether represent more than 4 the way for further research on the metabolic adaptation of an billion animals worldwide, are highly susceptible, and in this important facultative intracellular pathogen to target organs. livestock, the brucellae characteristically target the genital organs Author contributions: T.B., F.C., E.V.S., and J.-J.L. designed research; T.B., F.C., A.Z.-R., T.G., J.B., and cause abortion and infertility (1). Close to parturition, these and C.W. performed research; T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. analyzed 14 bacteria reach exceedingly high numbers (up to 10 bacteria per data; and T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. wrote the paper. conceptus) (2), thus making brucellosis abortion a highly conta- The authors declare no conflict of interest. gious condition. This article is a PNAS Direct Submission. It is accepted that this tropism and extensive multiplication relate 1T.B. and F.C. contributed equally to this work. to the high erythritol concentration in the genital organs of those 2E.V.S. and J.-J.L. contributed equally to this work. animals (3, 4). In vitro, this four-carbon polyol is a preferential car- 3To whom correspondence may be addressed. Email: [email protected] bon source of Brucella, promotes growth, and up-regulates the ex- or [email protected]. pression of major virulence factors and key enzymes of central This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metabolism (5–9). In vivo, it has been observed that parenteral 1073/pnas.1414622111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1414622111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17815–17820 Downloaded by guest on September 25, 2021 Fig. 1. Erythritol catabolism by Brucella.(A) The two first reactions of the “accepted” and the “revised” pathways are similar (highlighted in gray and further characterized in Figs. S1 and S2 and Table S1). Although it was previously admitted that L-3-tetrulose-4-P is afterward oxidized to dihydroxyacetone-phos- phate and CO2, we now show that this intermediate is converted to D-erythrose-4-P via three isomerization reactions catalyzed by EryC, TpiA2, and RpiB. The relevant enzyme-encoding genes are organized as illustrated in two operons also comprising two regulators (EryD and DeoR). (B) D-erythrose-4-P is further processed by enzymes of the pentose phosphate pathway to generate fructose-6-P and glyceraldehyde-3-P, two intermediates of glycolysis/gluconeogenesis (C). The proteins whose genes are induced by erythritol (9) are circled in red. The new proposed gene names are indicated in parentheses below the old designations. Fba, fructose bisphosphate aldolase; Fbp, fructose bisphosphatase; G6P, glucose-6-P; Gapdh, glyceraldehyde-3-P dehydrogenase; Pgk, phos- phoglycerate kinase; Pgm, phosphoglycerate mutase; R5P, ribose-5-P; Rpe, ribulose-5-P epimerase; Rpi, ribose-5-P isomerase; Ru5P, ribulose-5-P; S7P, sedo- heptulose-7-P; Tal, transaldolase; Tkt, transketolase; Tpi, triose-P isomerase; X5P, xylulose-5-P. Because the involvement of two putative isomerases is not sole carbon source requires isomerization of dihydroxyacetone-P considered in the accepted pathway, where a dehydrogenase and to glyceraldehyde-3-P, triose-P condensation by aldolase, and a decarboxylase are predicted to catalyze the last two steps, we dephosphorylation of fructose-1,6-P2 by fructose-1,6-bisphos- reassessed the erythritol pathway. A combination of genetic, phatase (FBPase). Brucella spp. carry only two FBPase genes [fbp biochemical, and isotopic evidence brought us to the conclusion and glpX (16)], and surprisingly, a double FBPase mutant (Bru- Δ Δ that erythritol metabolism involves three as-yet undescribed isom- cella abortus 2308 fbp glpx) showed no growth defect when provided with erythritol as the only carbon source (Fig. S3A), erization reactions catalyzed by EryC, TpiA2, and RpiB, which indicating that this polyol is converted not into trioses-P but into together lead to the formation of D-erythrose-4-P, rather than a product that can generate fructose-6-P without involvement
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