Cutting Edge: Human δγ T Cells Are Activated by Intermediates of the 2- C -methyl-d-erythritol 4-phosphate Pathway of Isoprenoid Biosynthesis This information is current as of September 24, 2021. Boran Altincicek, Jens Moll, Narciso Campos, Gesine Foerster, Ewald Beck, Jean-François Hoeffler, Catherine Grosdemange-Billiard, Manuel Rodríguez-Concepción, Michel Rohmer, Albert Boronat, Matthias Eberl and Hassan Jomaa Downloaded from J Immunol 2001; 166:3655-3658; ; doi: 10.4049/jimmunol.166.6.3655 http://www.jimmunol.org/content/166/6/3655 http://www.jimmunol.org/ References This article cites 49 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/166/6/3655.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! 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Print ISSN: 0022-1767 Online ISSN: 1550-6606. ● Cutting Edge: Human ␥␦ T Cells Are Activated by Intermediates of the 2-C- methyl-D-erythritol 4-phosphate Pathway of Isoprenoid Biosynthesis1 Boran Altincicek,2*† Jens Moll,† Narciso Campos,‡ Gesine Foerster,* Ewald Beck,† Jean-Franc¸ois Hoeffler,§ Catherine Grosdemange-Billiard,§ Manuel Rodrı´guez-Concepcio´n,‡ Michel Rohmer,§ Albert Boronat,‡ Matthias Eberl,† and Hassan Jomaa*† ficiently than IPP. Because of its structural resemblance with IPP, Activation of V␥9/V␦2 T cells by small nonprotein Ags is fre- FBPP is thought to be an intermediate of the 2-C-methyl-D-eryth- quently observed after infection with various viruses, bacteria, ritol 4-phosphate (MEP) pathway of IPP biosynthesis, which is Downloaded from and eukaryotic parasites. We suggested earlier that com- utilized by many pathogenic bacteria (16, 17) as well as protozoa pounds synthesized by the 2-C-methyl-D-erythritol 4-phos- harboring apicoplasts, such as Plasmodium falciparum (18), but phate (MEP) pathway of isopentenyl pyrophosphate synthesis apparently absent in vertebrates. However, the final proof of our are responsible for the V␥9/V␦2 T cell reactivity of many earlier suggestion that compounds synthesized by the MEP path- pathogens. Using genetically engineered Escherichia coli way are responsible for V␥9/V␦2 T cell reactivity of these infec- knockout strains, we now demonstrate that the ability of E. coli tious agents (9) has still been missing. To address this problem, we http://www.jimmunol.org/ extracts to stimulate ␥␦ T cell proliferation is abrogated when used different genetically engineered Escherichia coli strains to genes coding for essential enzymes of the MEP pathway, dxr or demonstrate that the ability of E. coli to stimulate ␥␦ T cell pro- gcpE, are disrupted or deleted from the bacterial liferation is abrogated when essential enzymes of the MEP path- genome. The Journal of Immunology, 2001, 166: 3655–3658. way are disrupted or deleted from the genome. The genome of wild-type (wt) E. coli contains the genes for the MEP pathway, of which dxs (coding for 1-deoxy-D-xylulose n humans, activation of ␥␦ T cells bearing the V␥9/V␦2 TCR 5-phosphate synthase, DOXP synthase, DXS) (19–21), and dxr by small nonprotein Ags is frequently observed after infec- (coding for DOXP reductoisomerase, DXR) (22, 23) have been tion with various viruses, bacteria, and eukaryotic parasites characterized in more detail. DXS and DXR catalyze the conden- by guest on September 24, 2021 I 3 (1–6). Although isopentenyl pyrophosphate (IPP) was the first sation of pyruvate with D-glyceraldehyde 3-phosphate to DOXP ligand described for V␥9/V␦2 T cells (7, 8), we have demonstrated and the subsequent formation of MEP, respectively. The gene that the natural amounts of IPP present in bacterial preparations do products of ygbP, ychB, and ygbB are involved in generating 2-C- not reach the minimum required for inducing T cell activation (9). methyl-D-erythritol 2,4-cyclopyrophosphate (MEcPP), with ␥ Recently, several other compounds were shown to stimulate V 9/ 4-diphosphocytidyl 2-C-methyl-D-erythritol (CDP-ME) as inter- V␦2 T cells, such as phosphorylated sugars, synthetic alkyl phos- mediate product (24–29). Most recently, an additional role for the phates, primary alkylamines, and 3-formyl-1-butyl pyrophosphate genes gcpE (30) and lytB (31) in the formation of IPP via the MEP (FBPP) (8, 10–15), the latter of which up to 1000-fold more ef- pathway was suggested (32, 33). Using molecular biological knockout techniques (34), we created E. coli strains deficient in *Jomaa Pharmaka GmbH, Giessen, Germany; †Biochemisches Institut, Justus-Liebig- dxr and gcpE, respectively, that utilize exogenously provided me- Universita¨t Giessen, Giessen, Germany; ‡Departament de Bioquı´mica i Biologı´a Mo- valonate (MVA) for IPP synthesis (33, 35) by complementation lecular, Universitat de Barcelona, Barcelona, Spain; and §Institut Le Bel, Universite´ with plasmids expressing the heterologous enzymes of the MVA Louis Pasteur/Centre National de la Recherche Scientifique, Strasbourg, France pathway (Fig. 1). In the present study, low molecular weight Received for publication November 30, 2000. Accepted for publication January ⌬ 22, 2001. (LMW) fractions from the parent E. coli strains as well as the dxr and ⌬gcpE strains were used for standard ␥␦ T cell stimulation The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance assays (9). with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grant 1999SGR 00032 from the Generalitat de Catalunya (to A.B.). Materials and Methods 2 Address correspondence and reprint requests to Dr. Boran Altincicek, Biochemis- Bacteria and plasmids ches Institut, Friedrichstrasse 24, D-35392 Giessen, Germany. E-mail address: Ϫ ⌬ [email protected] Construction of E. coli MC4100 (F araD139 (argF-lac)U169 relA1 rpsL150 flbB5301 strA thi deoC7 ptsF25) with a disruption in the dxr gene, 3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; CDP-ME, EcAB1-2, was published previously (35). EcAB1-2 bacteria were trans- 4-diphosphocytidyl 2-C-methyl-D-erythritol; DMAPP, dimethylallyl pyrophosphate; formed with plasmid pAB-M2 containing a synthetic operon to express the wt, wild type; DOXP, 1-deoxy-D-xylulose 5-phosphate; DXR, DOXP reductoisomer- ase; DXS, DOXP synthase; FBPP, 3-formyl-1-butyl pyrophosphate; ME, 2-C-methyl- coding region of Saccharomyces cerevisiae ERG12 (MVA kinase, MVK) D-erythritol; MEcPP, 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate; MEP, 2-C- and ERG19 (MVA pyrophosphate decarboxylase, MPD) genes and the methyl-D-erythritol 4-phosphate; MVA, mevalonate; MPD, MVA pyrophosphate human PMK cDNA (phosphomevalonate kinase, PMK) under the control decarboxylase; MVK, MVA kinase; PMK, phosphomevalonate kinase. of the arabinose-inducible PBAD promoter (35). MC4100 bacteria were Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 ● 3656 CUTTING EDGE Results and Discussion Incubation of human PBMC with LMW prepared from MC4100 bacteria stimulated expansion of ␥␦ T cells (Figs. 2 and 3). In contrast, no significant ␥␦ T cell reactivity was detected in the presence of LMW from EcAB1-2(pAB-M2) bacteria; this strain is mutated in the dxr gene while harboring plasmid pAB-M2 express- ing the three enzymes necessary for utilizing exogenously pro- vided MVA for IPP biosynthesis, MVK, PMK, and MPD (35). However, when grown on 2-C-methyl-D-erythritol (ME) instead of MVA, the capacity of EcAB1-2(pAB-M2) bacteria to stimulate ␥␦ T cells was partially restored. Differences between the control strain and dxr-deficient bacteria could be detected at LMW dilu- tions of down to 1 in 216; at this dilution, extracts from E. coli MC4100 exhibit a bioactivity which was comparable to the stim- ulation by IPP at 1.25 ␮M (Fig. 3). In addition to the laboratory strain MC4100, we engineered E. coli mutants on a wt genetic background, with complete in-frame gene deletions for either dxr or gcpE (33); these strains were com- plemented with plasmid pSC-MVA (Fig. 1). Not surprisingly, there was a significant increase in ␥␦ T cell numbers in the pres- Downloaded from ence of E. coli Ags (Fig. 4). Stimulation with LMW from wt E. FIGURE 1. Genetic and biochemical complementation of knockout E. coli and E. coli transformed with plasmid pSC-MVA, respectively, coli strains. E. coli cells were transfected with expression plasmids ␥␦ pAB-M2 or pSC-MVA, respectively, thus complementing knockout strains led to comparable T cell numbers. However, similar to EcAB1- ⌬ with the heterologous enzymes MVK, PMK, and MPD, for allowing 2(pAB-M2), LMW prepared from wt dxr(pSC-MVA) grown on MVA-dependent IPP synthesis. In wt E. coli, DOXP is synthesized from MVA did not induce marked ␥␦ T cell proliferation, thus implying pyruvate and D-glyceraldehyde 3-phosphate (GAP) by DXS and subse- an essential role for the MEP pathway in synthesizing potent ␥␦ T http://www.jimmunol.org/ quently modified to MEP by DXR. Growth of strains deficient in DXR can cell Ags. Furthermore, no response was observed with LMW from be restored by providing exogenous ME, which is then phosphorylated to wt⌬gcpE(pSC-MVA). Recently, we demonstrated that gcpE codes form MEP; the enzyme responsible for this step has not been identified yet.