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The Journal of Immunology, 2001, 166: 3655-3658.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Human {gamma}{delta} T Cells Are Activated by Intermediates of the 2-C-methyl-D-erythritol 4-phosphate Pathway of Isoprenoid Biosynthesis1

Boran Altincicek2,*,{dagger}, Jens Moll{dagger}, Narciso Campos{ddagger}, Gesine Foerster*, Ewald Beck{dagger}, Jean-François Hoeffler§, Catherine Grosdemange-Billiard§, Manuel Rodríguez-Concepción{ddagger}, Michel Rohmer§, Albert Boronat{ddagger}, Matthias Eberl{dagger} and Hassan Jomaa*,{dagger}

* Jomaa Pharmaka GmbH, Giessen, Germany; {dagger} Biochemisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany; {ddagger} Departament de Bioquímica i Biología Molecular, Universitat de Barcelona, Barcelona, Spain; and § Institut Le Bel, Université Louis Pasteur/Centre National de la Recherche Scientifique, Strasbourg, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Activation of V{gamma}9/V{delta}2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V{gamma}9/V{delta}2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate {gamma}{delta} T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In humans, activation of {gamma}{delta} T cells bearing the V{gamma}9/V{delta}2 TCR by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites (1, 2, 3, 4, 5, 6). Although isopentenyl pyrophosphate (IPP)3 was the first ligand described for V{gamma}9/V{delta}2 T cells (7, 8), we have demonstrated that the natural amounts of IPP present in bacterial preparations do not reach the minimum required for inducing T cell activation (9). Recently, several other compounds were shown to stimulate V{gamma}9/V{delta}2 T cells, such as phosphorylated sugars, synthetic alkyl phosphates, primary alkylamines, and 3-formyl-1-butyl pyrophosphate (FBPP) (8, 10, 11, 12, 13, 14, 15), the latter of which up to 1000-fold more efficiently than IPP. Because of its structural resemblance with IPP, FBPP is thought to be an intermediate of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of IPP biosynthesis, which is utilized by many pathogenic bacteria (16, 17) as well as protozoa harboring apicoplasts, such as Plasmodium falciparum (18), but apparently absent in vertebrates. However, the final proof of our earlier suggestion that compounds synthesized by the MEP pathway are responsible for V{gamma}9/V{delta}2 T cell reactivity of these infectious agents (9) has still been missing. To address this problem, we used different genetically engineered Escherichia coli strains to demonstrate that the ability of E. coli to stimulate {gamma}{delta} T cell proliferation is abrogated when essential enzymes of the MEP pathway 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 5-phosphate synthase, DOXP synthase, DXS) (19, 20, 21), and dxr (coding for DOXP reductoisomerase, DXR) (22, 23) have been characterized in more detail. DXS and DXR catalyze the condensation of pyruvate with D-glyceraldehyde 3-phosphate to DOXP and the subsequent formation of MEP, respectively. The gene products of ygbP, ychB, and ygbB are involved in generating 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate (MEcPP), with 4-diphosphocytidyl 2-C-methyl-D-erythritol (CDP-ME) as intermediate product (24, 25, 26, 27, 28, 29). Most recently, an additional role for the genes gcpE (30) and lytB (31) in the formation of IPP via the MEP pathway was suggested (32, 33). Using molecular biological knockout techniques (34), we created E. coli strains deficient in dxr and gcpE, respectively, that utilize exogenously provided mevalonate (MVA) for IPP synthesis (33, 35) by complementation with plasmids expressing the heterologous enzymes of the MVA pathway (Fig. 1Go). In the present study, low molecular weight (LMW) fractions from the parent E. coli strains as well as the {Delta}dxr and {Delta}gcpE strains were used for standard {gamma}{delta} T cell stimulation assays (9).



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  		FIGURE 1. Genetic and biochemical complementation of knockout E. coli strains. E. coli cells were transfected with expression plasmids pAB-M2 or pSC-MVA, respectively, thus complementing knockout strains with the heterologous enzymes MVK, PMK, and MPD, for allowing MVA-dependent IPP synthesis. In wt E. coli, DOXP is synthesized from pyruvate and D-glyceraldehyde 3-phosphate (GAP) by DXS and subsequently modified to MEP by DXR. Growth of strains deficient in DXR can be restored by providing exogenous ME, which is then phosphorylated to form MEP; the enzyme responsible for this step has not been identified yet. Finally, MEP is transformed into IPP via a largely unknown mechanism, with MEcPP and FBPP among putative intermediates, some of which represent potent {gamma}{delta} T cell stimuli.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacteria and plasmids

Construction of E. coli MC4100 (F- araD139 {Delta}(argF-lac)U169 relA1 rpsL150 flbB5301 strA thi deoC7 ptsF25) with a disruption in the dxr gene, EcAB1-2, was published previously (35). EcAB1-2 bacteria were transformed with plasmid pAB-M2 containing a synthetic operon to express the coding region of Saccharomyces cerevisiae ERG12 (MVA kinase, MVK) and ERG19 (MVA pyrophosphate decarboxylase, MPD) genes and the human PMK cDNA (phosphomevalonate kinase, PMK) under the control of the arabinose-inducible PBAD promoter (35). MC4100 bacteria were grown in 2xTYmedium, EcAB1-2(pAB-M2) in 2xTY medium supplemented with 100 µg/ml ampicillin, 6 µg/ml tetracycline, and 0.0004% L-arabinose, in the presence of either 1 mM MVA or 1 mM 2-C-methyl-D-erythritol (ME). E. coli wt{Delta}dxr and wt{Delta}gcpE with precise in-frame deletions 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{Delta}dxr and wt{Delta}gcpE in the presence of 150 µg/ml ampicillin and, where appropriate, 100 µM MVA. Bacteria were harvested from fresh liquid cultures at an OD at 600 nm of ~0.8, and LMW fractions were obtained as described using Amicon 3-kDa filters (Amicon, Witten, Germany) (9).

{gamma}{delta} 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 Pharmacia Biotech, Freiburg, Germany). Three x 105 PBMC/well were cultivated in RPMI 1640 medium supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 µg/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 für Klinische Immunologie und Transfusionsmedizin, Universität Giessen). LMW preparations were added at a dilution of 1 in 36:1 in 2196, corresponding to ~2.5 x 106-7.0 x 104 bacteria cells/well, respectively. Cells incubated with medium alone and cells stimulated with IPP at a concentration of 0.2–7.5 µM 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-CD3 and FITC-labeled pan-{gamma}{delta} mAbs (Becton Dickinson).

Statistical analysis

Data were expressed as mean ± SEM. Statistical analysis was performed using Student’s t test, with differences considered to be statistically significant at p < 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Incubation of human PBMC with LMW prepared from MC4100 bacteria stimulated expansion of {gamma}{delta} T cells (Figs. 2Go and 3Go). In contrast, no significant {gamma}{delta} 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 expressing the three enzymes necessary for utilizing exogenously provided 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 {gamma}{delta} T cells was partially restored. Differences between the control strain and dxr-deficient bacteria could be detected at LMW dilutions of down to 1 in 216; at this dilution, extracts from E. coli MC4100 exhibit a bioactivity which was comparable to the stimulation by IPP at 1.25 µM (Fig. 3Go).



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  		FIGURE 2. Disruption of the dxr gene abrogates the {gamma}{delta} T cell stimulatory potential of E. coli. PBMC were incubated with medium alone or with 1/36 dilutions of LMW prepared from E. coli MC4100, EcAB1-2(pAB-M2) grown on MVA, or EcAB1-2(pAB-M2) grown on ME and analyzed for {gamma}{delta} T cell outgrowth. The data represent means ± SEM from independently analyzed individuals (n = 4–6). Significant differences are indicated as *, p < 0.05.

 


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  		FIGURE 3. ME restores the {gamma}{delta} T cell stimulatory potential of E. coli with a disrupted dxr gene. PBMC were incubated with LMW prepared from E. coli MC4100 ({circ}), EcAB1-2(pAB-M2) grown on MVA ({triangleup}), or EcAB1-2(pAB-M2) grown on ME ({blacktriangleup}) and analyzed for {gamma}{delta} T cell outgrowth. A stock solution containing 270 µM IPP was used as control (•). The data represent typical results from several patients analyzed. Background values with medium alone in the experiment shown were at ~8%.

 
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 complemented with plasmid pSC-MVA (Fig. 1Go). Not surprisingly, there was a significant increase in {gamma}{delta} T cell numbers in the presence of E. coli Ags (Fig. 4Go). Stimulation with LMW from wt E. coli and E. coli transformed with plasmid pSC-MVA, respectively, led to comparable {gamma}{delta} T cell numbers. However, similar to EcAB1-2(pAB-M2), LMW prepared from wt{Delta}dxr(pSC-MVA) grown on MVA did not induce marked {gamma}{delta} T cell proliferation, thus implying an essential role for the MEP pathway in synthesizing potent {gamma}{delta} T cell Ags. Furthermore, no response was observed with LMW from wt{Delta}gcpE(pSC-MVA). Recently, we demonstrated that gcpE codes for an enzyme of the MEP pathway, catalyzing a reaction downstream of the formation of MEcPP (33).



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  		FIGURE 4. Both the dxr and the gcpE gene are needed for the {gamma}{delta} T cell stimulatory potential of E. coli. PBMC were incubated with medium alone or with 1/36 dilutions of LMW prepared from E. coli wt, wt(pSC-MVA), wt{Delta}dxr(pSC-MVA), or wt{Delta}gcpE(pSC-MVA) (all grown on MVA) and analyzed for {gamma}{delta} T cell outgrowth. The data represent means ± SEM from independently analyzed individuals (n = 4–6). Significant differences are indicated as *, p < 0.05.

 
Our data are in accordance with our previous prediction that metabolites of the MEP pathway are responsible for the {gamma}{delta} T cell reactivity to various pathogenic bacteria (9). Moreover, the fact that a mutation of the dxr gene almost totally abrogated the {gamma}{delta} T cell expansion observed with extracts from E. coli MC4100 and wt E. coli suggests that intermediates of the MEP pathway that downstream the formation of CDP-ME (or metabolites of these intermediates) may be the most important nonprotein Ags eliciting a {gamma}{delta} T cell response to these bacteria; DOXP, MEP, and CDP-ME do not activate human {gamma}{delta} T cells (Ref. 9 ; our unpublished observations). This conclusion is supported by recent findings of Belmant et al. (12), who identified FBPP as potent {gamma}{delta} T cell Ag and suggested that it may represent a late metabolite of the MEP pathway. Most recently, we presented genetic evidence that branching in the MEP pathway results in separate synthesis of IPP and dimethylallyl pyrophosphate (DMAPP) (35); additionally, IPP and DMAPP can be interconverted by the enzyme IPP isomerase. As the {Delta}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 {gamma}{delta} T cells. A great variety of pathogens utilize this pathway for biosynthesis of isoprenoids (36, 37, 38). Thus, the unconventional {gamma}{delta} T cell reactivity to common LMW compounds ensures a quick and efficient cellular immune response to a broad range of evolutionarily distant pathogens that may otherwise escape classical MHC-restricted mechanisms (39). However, it is clear that other organic compounds may be of relevance in some bacterial infections, as Bukowski et al. (13) showed that {gamma}{delta} T cell stimulation by Proteus morganii extracts was not affected by prior alkaline phosphatase treatment, but was due to nonphosphorylated alkylamines. Activation of human V{gamma}9/V{delta}2 T cells by phosphoantigens leads to clonal expansion, enhanced cytotoxicity, secretion of proinflammatory cytokines, expression of C-C chemokines, and up-regulation of chemokine receptors (5, 40, 41, 42), and is therefore crucial for regulating the immune response in a wide range of bacterial infections (43, 44, 45, 46, 47). However, the fact that many {gamma}{delta} T cell-activating pathogens are capable of establishing chronic and debilitating diseases, such as tuberculosis and malaria, implies that in those infections, stimulation of {gamma}{delta} T cells may represent a potent immune evasion strategy. In fact, there is some evidence that {gamma}{delta} T cells can down-regulate specific immune responses and/or induce tolerance (6, 48, 49, 50). Thus, in addition to its value as drug target for the treatment of various bacterial infections and malaria (18, 21), the MEP pathway may be of increasing interest for the future development of immunomodulatory agents.


   Acknowledgments
 
We thank the board of directors of the Academic Hospital Center of the University of Giessen for their generous support. We gratefully acknowledge 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.


   Footnotes
 
1 This work was supported in part by Grant 1999SGR 00032 from the Generalitat de Catalunya (to A.B.). Back

2 Address correspondence and reprint requests to Dr. Boran Altincicek, Biochemisches Institut, Friedrichstrasse 24, D-35392 Giessen, Germany. Back

3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; CDP-ME, 4-diphosphocytidyl 2-C-methyl-D-erythritol; DMAPP, dimethylallyl pyrophosphate; wt, wild type; DOXP, 1-deoxy-D-xylulose 5-phosphate; DXR, DOXP reductoisomerase; DXS, DOXP synthase; FBPP, 3-formyl-1-butyl pyrophosphate; ME, 2-C-methyl-D-erythritol; MEcPP, 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; MPD, MVA pyrophosphate decarboxylase; MVK, MVA kinase; PMK, phosphomevalonate kinase. Back

Received for publication November 30, 2000. Accepted for publication January 22, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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