Dirk A. E. Dobbelaere

The top 100 questions of importance to the future of global agriculture

Despite a significant growth in food production over the past half-century, one of the most important challenges facing society today is how to feed an expected population of some nine billion by the middle of the 20th century. To meet the expected demand for food without significant increases in prices, it has been estimated that we need to produce 70–100 per cent more food, in light of the growing impacts of climate change, concerns over energy security, regional dietary shifts and the Millennium Development target of halving world poverty and hunger by 2015. The goal for the agricultural sector is no longer simply to maximize productivity, but to optimize across a far more complex landscape of production, rural development, environmental, social justice and food consumption outcomes. However, there remain significant challenges to developing national and international policies that support the wide emergence of more sustainable forms of land use and efficient agricultural production. The lack of information flow between scientists, practitioners and policy makers is known to exacerbate the difficulties, despite increased emphasis upon evidence-based policy. In this paper, we seek to improve dialogue and understanding between agricultural research and policy by identifying the 100 most important questions for global agriculture. These have been compiled using a horizon-scanning approach with leading experts and representatives of major agricultural organizations worldwide. The aim is to use sound scientific evidence to inform decision making and guide policy makers in the future direction of agricultural research priorities and policy support. If addressed, we anticipate that these questions will have a significant impact on global agricultural practices worldwide, while improving the synergy between agricultural policy, practice and research. This research forms part of the UK Governments Foresight Global Food and Farming Futures project.

A Metabolic Enzyme as a Primary Virulence Factor of Mycoplasma mycoides subsp. mycoides Small Colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes.

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

Macrophage-parasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata.

Theileria annulata is a tick‐transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mɸ) or B cell lineage. Bone marrow cells, bone marrow cell‐derived, and monocyte‐derived Mɸ were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class‐I and II, CD44, CD45, and the myeloid marker DH598‐surface markers CD14, CD11b, M‐M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down‐regulated. Likewise, transformed cells failed to express Mɸ functions (Fc‐receptor‐mediated phagocytosis, phorbol myristate acetate‐induced oxidative burst, lipopolysaccharide‐induced tumor necrosis factor α, and nitric oxide generation and procoagulant activity up‐regulation). Mɸ origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the reacquisition of monocyte lineage properties, as evidenced by up‐regulation of CD14, and by re‐acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mɸ transformed by T. annulaia appear to undergo a process of parasite‐induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite. J. Leukoc. Biol. 61: 459–468; 1997.

Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas Ligand-Induced Apoptosis

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-xL are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3‐K‐generated phosphoinositides, in Theileria‐transformed leucocytes. Akt/PKB is constitutively activated in a PI3‐K‐ and parasite‐dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF‐κB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3‐K‐Akt/PKB pathway and NF‐κB activation, resulting in protection against apoptosis. In Theileria‐transformed leucocytes, activation of the NF‐κB and the PI3‐K‐Akt/PKB pathways are not directly linked. The PI3‐K‐Akt/PKB pathway does not contribute to the persistent induction of IκBα phosphorylation, NF‐κB DNA‐binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF‐κB‐dependent protection against apoptosis is not dependent on a functional PI3‐K‐Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria‐transformed T cells.

The Transforming Parasite Theileria Co-opts Host Cell Mitotic and Central Spindles to Persist in Continuously Dividing Cells

The transforming protozoan Theileria recruits Plk1, a host kinase that regulates mitosis, to its surface and engages spindle microtubules to secure its division and inheritance into both daughter cells.

Theileria parva: taking control of host cell proliferation and survival mechanisms.

The intracellular parasite Theileria parva infects and transforms bovine T‐cells, inducing their uncontrolled proliferation and spread in non‐lymphoid as well as lymphoid tissues. This parasite‐induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva‐transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva‐infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T‐cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen‐activated protein kinase (MAPK), NF‐κB and phosphoinositide‐3‐kinase (PI3‐K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP‐1 and ATF‐2. A crucial step in the transformation process is the persistent activation of the transcription factor NF‐κB, which protects T. parva‐transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI‐3K, in the continuous proliferation of T. parva‐transformed lymphocytes.

N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

The serine protease inhibitor N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-κB activation in T. parva-transformed T-cells, with phosphorylation of IκBα and IκBβ being inhibited with different kinetics. TPCK-mediated inhibition of IκB phosphorylation, NF-κB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-κB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-κB activation pathway(s).

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.