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Isoprenoid Biosynthesis This Information Is Current As of September 24, 2021

Isoprenoid Biosynthesis This Information Is Current As of September 24, 2021

Cutting Edge: Human δγ T Cells Are Activated by Intermediates of the 2- C -methyl-d-erythritol 4-phosphate Pathway of Isoprenoid 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/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. 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, 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 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 responsible for this step has not been identified yet. for an enzyme of the MEP pathway, catalyzing a reaction down- Finally, MEP is transformed into IPP via a largely unknown mechanism, stream of the formation of MEcPP (33). with MEcPP and FBPP among putative intermediates, some of which rep- Our data are in accordance with our previous prediction that resent potent ␥␦ T cell stimuli. metabolites of the MEP pathway are responsible for the ␥␦ T cell reactivity to various pathogenic bacteria (9). Moreover, the fact ␥␦ grown in 2ϫTYmedium, EcAB1-2(pAB-M2) in 2ϫTY medium supple- that a mutation of the dxr gene almost totally abrogated the T mented with 100 ␮g/ml ampicillin, 6 ␮g/ml tetracycline, and 0.0004% by guest on September 24, 2021 L-arabinose, in the presence of either 1 mM MVA or 1 mM 2-C-methyl- D-erythritol (ME). E. coli wt⌬dxr and wt⌬gcpE with precise in-frame de- letions of dxr and gcpE, respectively, derived from wt K-12 strain DSM no. 498, ATCC 23716, and plasmid pSC-MVA with a synthetic operon to express S. cerevisiae ERG12 (MVK), ERG8 (PMK), and ERG19 (MPD) were described elsewhere (33). E. coli wt strains were grown in standard 1 medium (Merck, West Point, PA), wt⌬dxr and wt⌬gcpE in the presence of 150 ␮g/ml ampicillin and, where appropriate, 100 ␮M MVA. Bacteria were harvested from fresh liquid cultures at an OD at 600 nm of ϳ0.8, and LMW fractions were obtained as described using Amicon 3-kDa filters (Amicon, Witten, Germany) (9). ␥␦ T cell stimulation assays Stimulation assays were performed as described previously (9). In brief, PBMC from healthy donors were isolated from heparinized peripheral blood by density centrifugation over Ficoll-Hypaque (Amersham Pharma- cia Biotech, Freiburg, Germany). Three ϫ 105 PBMC/well were cultivated in RPMI 1640 medium supplemented with 25 mM HEPES, 2 mM L-glu- tamine, 100 ␮g/ml penicillin-streptomycin, 100 U/ml recombinant human IL-2 (all from Life Technologies, Karlsruhe, Germany), and 10% pooled human AB serum (kindly provided by the Institut fu¨r Klinische Immu- nologie und Transfusionsmedizin, Universita¨t Giessen). LMW prepara- tions were added at a dilution of 1 in 36:1 in 2196, corresponding to ϳ2.5 ϫ 106-7.0 ϫ 104 bacteria cells/well, respectively. Cells incubated with medium alone and cells stimulated with IPP at a concentration of 0.2–7.5 ␮M served as negative and positive controls, respectively. Cells were harvested on day 7 and analyzed on a FACSCalibur supported with CellQuest (Becton Dickinson, Heidelberg, Germany) using PE-labeled anti- FIGURE 2. Disruption of the dxr gene abrogates the ␥␦ T cell stimu- CD3 and FITC-labeled pan-␥␦ mAbs (Becton Dickinson). latory potential of E. coli. PBMC were incubated with medium alone or with 1/36 dilutions of LMW prepared from E. coli MC4100, EcAB1- Statistical analysis 2(pAB-M2) grown on MVA, or EcAB1-2(pAB-M2) grown on ME and Data were expressed as mean Ϯ SEM. Statistical analysis was performed analyzed for ␥␦ T cell outgrowth. The data represent means Ϯ SEM from using Student’s t test, with differences considered to be statistically signif- independently analyzed individuals (n ϭ 4–6). Significant differences are .p Ͻ 0.05 ,ء icant at p Ͻ 0.05. indicated as The Journal of Immunology 3657

can be interconverted by the enzyme IPP isomerase. As the ⌬gcpE mutation is lethal in E. coli, we conclude that the step catalyzed by GcpE is located before the branch point, which has been proposed to be controlled by LytB (32). Taken together, our data clearly demonstrate that compounds synthesized as intermediates or, which is also conceivable, as side products of the MEP pathway activate human ␥␦ T cells. A great variety of pathogens utilize this pathway for biosynthesis of iso- prenoids (36–38). Thus, the unconventional ␥␦ T cell reactivity to common LMW compounds ensures a quick and efficient cellular immune response to a broad range of evolutionarily distant patho- gens that may otherwise escape classical MHC-restricted mecha- nisms (39). However, it is clear that other organic compounds may be of relevance in some bacterial infections, as Bukowski et al. ␥␦ FIGURE 3. ME restores the T cell stimulatory potential of E. coli (13) showed that ␥␦ T cell stimulation by Proteus morganii ex- with a disrupted dxr gene. PBMC were incubated with LMW prepared tracts was not affected by prior alkaline phosphatase treatment, but from E. coli MC4100 (E), EcAB1-2(pAB-M2) grown on MVA (‚), or Œ ␥␦ was due to nonphosphorylated alkylamines. Activation of human EcAB1-2(pAB-M2) grown on ME ( ) and analyzed for T cell out- ␥ ␦ growth. A stock solution containing 270 ␮M IPP was used as control (F). V 9/V 2 T cells by phosphoantigens leads to clonal expansion, The data represent typical results from several patients analyzed. Back- enhanced cytotoxicity, secretion of proinflammatory cytokines, ex- ground values with medium alone in the experiment shown were at ϳ8%. pression of C-C chemokines, and up-regulation of chemokine re- Downloaded from ceptors (5, 40–42), and is therefore crucial for regulating the im- mune response in a wide range of bacterial infections (43–47). ␥␦ cell expansion observed with extracts from E. coli MC4100 and wt However, the fact that many T cell-activating pathogens are E. coli suggests that intermediates of the MEP pathway that down- capable of establishing chronic and debilitating diseases, such as stream the formation of CDP-ME (or metabolites of these inter- tuberculosis and malaria, implies that in those infections, stimula- ␥␦ mediates) may be the most important nonprotein Ags eliciting a ␥␦ tion of T cells may represent a potent immune evasion strategy. http://www.jimmunol.org/ ␥␦ T cell response to these bacteria; DOXP, MEP, and CDP-ME do In fact, there is some evidence that T cells can down-regulate not activate human ␥␦ T cells (Ref. 9; our unpublished observa- specific immune responses and/or induce tolerance (6, 48–50). tions). This conclusion is supported by recent findings of Belmant Thus, in addition to its value as drug target for the treatment of et al. (12), who identified FBPP as potent ␥␦ T cell Ag and sug- various bacterial infections and malaria (18, 21), the MEP pathway gested that it may represent a late metabolite of the MEP pathway. may be of increasing interest for the future development of im- Most recently, we presented genetic evidence that branching in the munomodulatory agents. MEP pathway results in separate synthesis of IPP and dimethyl- allyl pyrophosphate (DMAPP) (35); additionally, IPP and DMAPP Acknowledgments We thank the board of directors of the Academic Hospital Center of the by guest on September 24, 2021 University of Giessen for their generous support. We gratefully acknowl- edge Martin Hintz, Ann-Kristin Kollas, Silke Sanderbrand, and Jochen Wiesner for their help and their stimulating discussion; Irina Steinbrecher, Dajana Henschker, and Ursula Jost for technical assistance; and Gergis Bassili for taking our blood.

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