Armin Reichenberg

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human γδ T cells in Escherichia coli

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 ΔlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1H, 13C and 31P NMR spectroscopy, and NOESY analysis as (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate (HMB‐PP). HMB‐PP is 104 times more potent in activating human Vγ9/Vδ2 T cells than isopentenyl pyrophosphate.

Microbial isoprenoid biosynthesis and human γδ T cell activation

Human Vγ9/Vδ2 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 identification of the specific activator was only possible due to recent success in a seemingly remote field: 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 Vγ9/Vδ2 T cell activator known, with an EC50 of 0.1 nM.

LytB protein catalyzes the terminal step of the 2‐C‐methyl‐D‐erythritol‐4‐phosphate pathway of isoprenoid biosynthesis

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady‐state kinetic parameters of LytB protein. The maximal specific activity of 6.6±0.3 μmol min−1 mg−1 protein was determined at pH 7.5 and 60°C. The k cat value of the LytB protein was 3.7±0.2 s−1 and the K m value for HMBPP was 590±60 μM.

Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis

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 purified under dioxygen‐free conditions. The protein was enzymatically active in converting 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate (MEcPP) into (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min−1 mg−1 at pH 7.5 and 55°C. The k cat value was 0.4 s−1 and the K m value for HMBPP 0.42 mM.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid‐like organelle (apicoplast) by the mevalonate independent 1‐deoxy‐d‐xylulose‐5‐phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe–4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin‐NADP+ reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin‐NADP+ reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

Diaryl ester prodrugs of fR900098 with improved in vivo antimalarial activity

The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.

Prolonged (E)-4-Hydroxy-3-Methyl-But-2-Enyl Pyrophosphate-Driven Antimicrobial and Cytotoxic Responses of Pulmonary and Systemic Vγ2Vδ2 T Cells in Macaques

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.

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity.

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.

Accumulation of a potent gammadelta T-cell stimulator after deletion of the lytB gene in Escherichia coli.

Activation of human Vγ9/Vδ2 T cells by many pathogens depends on the presence of small phosphorylated non‐peptide compounds derived from the 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway of isoprenoid biosynthesis. We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vγ9/Vδ2 T‐cell activator accumulates by a factor of approximately 150 compared to wild‐type E. coli. The compound responsible for the strong immunogenicity of this E. coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway. Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB‐deficient E. coli mutant led to upregulation of T‐cell activation markers on the surface of Vγ9/Vδ2 T cells as well as proliferation and expansion of Vγ9/Vδ2 T cells. This response was dependent on costimulatory growth factors, such as interleukin (IL)‐2, IL‐15 and IL‐21. Significant levels of interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were secreted in the presence of IL‐2 and IL‐15, but not in the presence of IL‐21, demonstrating that proliferating phosphoantigen‐reactive Vγ9/Vδ2 T cells do not necessarily produce proinflammatory cytokines.

Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways

Background Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. Methodology/Principal Findings Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. Conclusions/Significance Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.