Ann-Kristin Kollas

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.

LytB, a novel gene of the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway of isoprenoid biosynthesis in Escherichia coli

The mevalonate‐independent 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

GcpE Is Involved in the 2-C-Methyl-d-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.

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.

The interplay between classical and alternative isoprenoid biosynthesis controls γδ T cell bioactivity of Listeria monocytogenes

Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non‐mevalonate, 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway. The latter is found in many microbial pathogens and proceeds via (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate (HMB‐PP), a potent activator of human Vγ9/Vδ2 T cells. Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently. Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability. Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway. Vγ9/Vδ2 T cell bioactivity is increased in ΔlytB mutants where HMB‐PP accumulation is expected, and lost in ΔgcpE mutants which fail to produce HMB‐PP.