The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli v vlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1 H, 13 C and 31 P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10 4 times more potent in activating human VQ Q9/VN N2 T cells than isopentenyl pyrophosphate. ß

Human VQ Q9/VN N2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non-peptide antigens has been enigmatic until recently. The break-through in identi¢cation of the speci¢c activator was only possible due to recent success in a seemingly remote ¢eld: the elucidation of the reaction steps of the newly discovered 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) (HMB-PP), turned out to be by far the most potent VQ Q9/VN N2 T cell activator known, with an EC 50 of 0.1 nM. ß

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was puri¢ed to apparent homogeneity. The puri¢ed LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a de¢ned in vitro system. The reaction products were identi¢ed as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal speci¢c activity of 6.6 þ 0.3 W Wmol min 31 mg 31 protein was determined at pH 7.5 and 60 ‡C. The k cat value of the LytB protein was 3.7 þ 0.2 s 31 and the K m value for HMBPP was 590 þ 60 W WM.

Although phosphoantigen-specific Vγ2Vδ2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these γδ T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vγ2Vδ2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vγ2Vδ2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3–4 mo, although circulating Vγ2Vδ2 T cells had returned to baseline levels weeks prior. The Vγ2Vδ2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7−CD45RA−CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-γ up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vγ2Vδ2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ αβ T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vγ2− T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vγ2Vδ2 T cells may confer immunotherapeutics against infectious diseases and cancers.

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and puri¢ed under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-Derythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal speci¢c activity was 0.6 W Wmol min 31 mg 31 at pH 7.5 and 55 ‡C. The k cat value was 0.4 s 31 and the K m value for HMBPP 0.42 mM.