Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-D-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/ApppI that directly stimulate. In this study, we further characterize stimulation by these compounds, and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates. However, the continuous presence of aminobisphosphonates was toxic for T cells, and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known antigen presenting molecules, and resistant to fixation. New classes of stimulatory compounds–mevalonate, the alcohol of HMBPP, and alkenyl phosphonates–likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct antigens. Transfection of APC with siRNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells, and increased cellular IPP. siRNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.

Human Vgamma2Vdelta2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vgamma2Vdelta2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vgamma2Vdelta2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vgamma2Vdelta2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vgamma2Vdelta2 T cells in vivo upon engraftment into severe combined immunodeficiency-beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vgamma2Vdelta2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vgamma2Vdelta2 T cells to respond to a broad array of pathogens using this pathway.

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The “red tide” organism Karenia brevis (Davis) Hansen & Moestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ8(14) sterols. The two predominant sterols, (24R)‐4α‐methylergosta‐8(14), 22‐dienol and (24R)‐4α‐methyl‐27‐norergosta‐8(14), 22‐dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non‐nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, ⌬ 5,7 sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S -adenosyl-L -methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form ⌬ 22 sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen. The lanosterol derivatives, 24-methylenelanost-8-en-3 ␤ -ol and ( Z )-24-ethylidenelanost-8-en-3 ␤ -ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci , were also detected among the sterols of the rat-derived P. carinii organisms. Pneumocystis pneumonia (PcP) remains among the most prevalent opportunistic infections in immunocompromised individuals such as AIDS patients. It has becoming evident that the high incidence of PcP in AIDS patients is global and that the organism can infect other immunodeficient people such as patients undergoing chemotherapy for cancer or solid organ transplant (1, 2), children prior to becoming fully immunocompetent, and the elderly with diminished immune systems. Pneumocystis can also transiently colonize normal, healthy people and animals without causing overt symptoms of respiratory disorder. The combination of trimethoprim and sulfamethoxazole and other agents (e.g., pentamidine, atovaquone) used for prophylaxis and for clearing PcP has successfully reduced the number of deaths directly attributed to PcP. However, AIDS and other patients with prolonged immunodeficiency experience recurrent Pneumocystis jiroveci ( Pneumocystis carinii f. sp. hominis ) (3, 4) infections. Also, some individuals cannot tolerate these drugs and suffer undesirable side effects. The development of drug-resistant pathogen populations is of serious concern, making it imperative to develop a larger armamentarium of diverse drugs to circumvent these problems (5 Ϫ 7). Although the organism can be maintained in long-term, small-volume axenic cultures (8), growth is very slow and insufficient numbers of organisms are obtained for most biochemical studies. Thus, Pneumocystis remains an organism considered difficult to manipulate for experimental work. Despite this difficulty, much is now known about the organism's lipids from analyses performed on preparations purified from infected rat lungs (9 Ϫ 11...

Biosynthetic studies on neocarzinostatin chromophore A ( N C S Chrom A) were carried out on the basis of the incorporation of singly and doubly I3C labeled acetate precursors as well as radiolabeled [methyl-3H]methionine, ['4C]sodium bicarbonate, and ['4C]sodium acetate by cultures of Streptomyces carzinostaticus ( A T C C # 15944 F-42). The results suggest that the N-methyl of the fucosamine and the 0-methyl of the naphthoic acid moieties a r e derived from methionine via S-adenosylmethionine and the cyclic carbonate carbonyl carbon from carbonate. T h e acetate incorporation results show that

Salvinorin A, a neoclerodane diterpenoid, isolated from the Mexican hallucinogenic plant Salvia divinorum, is a potent kappa-opioid receptor agonist. Its biosynthetic route was studied by NMR and HR-ESI-MS analysis of the products of the incorporation of [1-(13)C]-glucose, [Me-(13)C]-methionine, and [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose into its structure. While the use of cuttings and direct-stem injection were unsuccessful, incorporation of (13)C into salvinorin A was achieved using in vitro sterile culture of microshoots. NMR spectroscopic analysis of salvinorin A (2.7 mg) isolated from 200 microshoots grown in the presence of [1-(13)C]-glucose established that this pharmacologically important diterpene is biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway, instead of the classic mevalonic acid pathway. This was confirmed further in plants grown in the presence of [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose. In addition, analysis of salvinorin A produced by plants grown in the presence of [Me-(13)C]-methionine indicates that methylation of the C-4 carboxyl group is catalyzed by a type III S-adenosyl-L-methionine-dependent O-methyltransferase.