Spring Meeting - University of Reading,
29th - 31st March 1999

Plenary Lecture: The evolution of trypanosomes

Wendy Gibson. School of Biological Sciences, University of Bristol.

Trypanosomes are parasites of all vertebrate classes. So far only Trypanosoma brucei has been shown to undergo some form of sexual reproduction and this lab has spent much time trying to elucidate the mechanism of genetic exchange in this trypanosome. Genetic exchange takes place in the vector, in which T. brucei unfortunately has a very complex lifecycle. With a view to finding relatives of T. brucei likely to undergo genetic exchange, but with simpler lifecycles, we constructed phylogenetic trees for trypanosomes, based on 18S ribosomal DNA sequences. Close relatives of T. brucei were not apparent from the tree, other than the other tsetse-transmitted trypanosomes, but we were able to elucidate the evolutionary relationships between various vertebrate trypanosomes, including the pathogens T. brucei and T. cruzi. The 18S tree indicated a monophyletic origin for genus Trypanosoma within the Kinetoplastida, using Trypanoplasma borreli as the outgroup. The African mammalian tsetse-transmitted trypanosomes comprised a separate clade, which appears to have diverged at an early date. Thus, T. brucei and T. cruzi, although both now human pathogens, had ancient and divergent origins. We have attempted to date the time of divergence of the African clade from paleogeographical evidence, which suggests the mid-Cretaceous or 100 million years ago, when Africa separated from the rest of the continental land masses. Besides adaptation to a tsetse vector, with concomitant transmission via saliva, these trypanosome species share the characteristic of antigenic variation. Trypanosoma cruzi and relatives form a second clade, with a possible southern supercontinent evolutionary origin. This has implications for the evolution of trypanosome species of Australian mammals. Trypanosome species from fish and amphibia form a third distinct clade; these species are linked by their dependence on aquatic leeches for transmission.

The induction of host cell genes by Theileria parasites

-Theileria I have known

Duncan Brown. CTVM, R(D)SVS, The University of Edinburgh.

A superficial account is given of personal involvement in research into tick-borne diseases over some 40+ years. This research was directed at solving animal health problems caused primarily by three pathogenic Theileria of domestic ruminants - Theileria parva and T.annulata of cattle and T.lestoquardi (= T.hirci) of sheep and goats - and covered much of what is known as the 'old world'. A first aquaintance with East Coast fever (ECF) caused by T.parva in Kenya, in 1956, showed there was much to be done. The disease killed over 90% of infected susceptible taurine (European) cattle if infected despite treatment with either intravenous turpentine or oral Dettol. Prevention by draconican application of acaricides to break the tick transmission cycle was the only means of control. There was need for both treatment of the clinical disease and a vaccine to prevent it. Standing firmly on the shoulders of those that had gone before, these problems were tackled, in the process of which many other questions emerged - a few of which have been answered by sequential teams of African and more exotic scientists.

Firstly, additional chemotherapeutics were developed and screened - in vivo and in vitro - to complement the tetracyclines which were prophylactic and the 8-aminoquinolines - which killed the merozoite / piroplasm and blocked infection of the tick. These new drugs - naphthoquinones and quinazolinones - were screened and found to be active against the macroschizont in in vitro culture. This culture, which could be established by in vitro infection of bovine mononuclear cells with sporozoites harvested from ticks led the way to other steps forward. The vertebrate life cycle - from sporozoite to merozoite - could now be established in vitro and stage-specific immune mechanisms studied. While sporozoites of T.parva preferentially infected and transformed T lymphocytes, those of T.annulata and T.lestoquardi infected monocyte / macrophages or B cells of their respective species. This selective affinity explains much about both the immune mechanisms which operate against these parasites and the methods used to vaccinate against these diseases. The parasite, on invading its target cell, transformed and immortalised it - by a mechanism which is the subject of this workshop. This immortalisation is both exciting and unusual, though not unique amongst apicomplexan parasites.

A similar symbiosis between protozoan and host cell exists in Glugea of stickleback, Haemogregarina of turbot and Eimeria crandallis of sheep. These provide fascinating tools to work with and also, for T.annulata and T.hirci, live vaccines comprising attenuated macroschizont-infected cell lines.

For T.parva, however, cattle could not be infected or immunised with infected, allogeneic cell lines. The only way to infect cattle with T.parva and protect them against ECF is with sporozoites from the tick. This has meant that the complete parasite lifecycle must be maintained. This, in turn, has necessitated detailed study of stage-specific antigens and the immune responses to them. We are now at a point where candidate recombinant antigens of the sporozoite, the merozoite and, ultimately and most difficult, the schizont are being evaluated in the laboratory and the field. They are in competition with two vaccines which are unique examples of live protozoan vaccines which though cumbersome, provide effective immunity: live, attenuated cell culture (macroschizont) vaccines for T.annulata and T.lestoquardi (hirci) and infection, using sporozoites harvested from infected ticks and cryopreserved as stabilates, and treatment for T.parva.

An upstream element of the Tams1 gene is a putative site for regulation of transcription during differentiation to the merozoite in Theileria annulata

Brian Shiels, Sue McKellar, Mark Fox, Kim Lyons, Jane Kinnaird and David Swan. University of Glasgow.

Previous studies have found that the gene, Tams1, encoding the major merozoite piroplasm surface antigen (mMPSA) is expressed in the preceding stage of the life cycle, the macroschizont. It was proposed that this was due to the presence of regulators of merozoite gene expression in the macroschizont, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run on analysis showed that Tams1 expression is controlled, at least in part, at the transcriptional level. Mapping of the transcriptional start site showed it had homology with the consensus sequence of the eukaryotic initiator (Inr) motif. In addition, comparison of 5' flanking sequences of Tams1, Tpms1 (T.parva) and Tsms1 (T. sergenti) revealed three regions of homology. Study of the Tams1 upstream region by the electrophoretic mobility shift assay (EMSA) demonstrated that a 23 bp motif (CAT1) specifically bound factors in extracts of parasite enriched nuclear fractions. Mutagenesis of this motif defined the consensus binding site to a 9 bp stretch: 5'-TTTGTAGGG-3'. EMSA using nuclear extracts from differentiation time course points, and quantification of the level of Tams1 5' DNA template in parasite nuclear fractions, showed that CAT1 binding factors are present in the macroschizont and that the level of these factors increase relative to their DNA template. The demonstration that 1 kb of the Tams1 5' region (including CAT1 and Inr) confers transcription of a G free cassette construct in vitro will allow future studies to functionally determine the role of these motifs in regulating Tams1 transcription.

CDKs: The why and the wherefore in the Theileria life cycle.

Jane H. Kinnaird, Mary Logan, Geoffrey Ingram, Gordon Langsley* and Brian Shiels. Dept of Veterinary Parasitology, University of Glasgow. *Unit of Experimental Parasitology, Institut Pasteur, Paris.

Parasite multiplication and life cycle stage transitions are intimately associated with pathogenesis. Our aim is to understand the regulatory mechanisms involved. One means by which regulation of a wide variety of basic cellular processes can be carried out is by protein phosphorylation. The cyclin dependent kinase group (CDKs) are ubiquitous, highly conserved serine/threonine protein kinases some of which are involved in regulating progression of the cell cycle, although others have roles in cell cycle entry and in differentiation. Two members of the family have been characterised from Theileria annulata. The first, ThaCRK2 encodes a 34 kDa polypeptide, closely related to CDK1/2 which are known to govern cell cycle progression. ThaCRK2 is present in all dividing life cycle stages, consistent with a role in parasite division. IFAT has shown that ThaCRK2 localises to the macroschizont cytoplasm, with a transitory increase in reactivity late in merozoite formation. In T. annulata the piroplasm stage does not show extensive multiplication but high levels of ThaCRK2 are present compared to other markers of nuclear division. Therefore, ThaCRK2 could have other roles outwith parasite division. The extremely high level of conservation of this CDK within the Apicomplexa points to a pivotal role in the life cycle. The second, ThaCRK3 encodes a 45 kDa polypeptide with most significant homology to the CDK7 class, associated with activation of other CDKs. A system has been developed to affinity purify kinases from native piroplasm extracts. At least two activities have been identified in this way, one of which is attributable to ThaCRK2 confirming its activity in the piroplasm stage. It is possible that either or both of these kinases will be implicated in regulation, by phosphorylation, of molecules involved in parasite-host interaction. We also consider changes in expression of a range of host and parasite replication markers including bovine CDK1 and ribonucleotide reductase, during differentiation of the macroschizont to the extracellular merozoite.

Matrix metalloproteinases, metastasis macroschizonts, mutagenesis and motifs. MMMMMM!!!

Rachel Adamson and Roger Hall. Department of Biology, University of York .

Infection of bovine leucocytes with the transforming parasite Theileria annulata, results in the de novo synthesis of host matrix metalloproteinases. These enzymes have been shown to be involved in the metastasis and dissemination of infected cells in both in vitro invasion assays and parasite dissemination in scid mice. One of these enzymes has been characterised in detail and is the bovine homologue of human matrix metalloproteinase 9 (MMP9). We have previously demonstrated that transcriptionof this gene is upregulated by the macroschizont stage of T. annulata. Recently, we have isolated and characterised the 5' upstream region of a genomic copy of this gene and we are currently assessing promoter function in transfection assays using the CAT reporter gene. The transcription factors AP-1 and NFKB play important regulatory roles in MMP transcription and we have observed the upregulation of both AP-1 and NFKB activities in infected cells. Both NFKB and AP-1 regulatory sequences are present in the 5' sequence of the MMP9 gene and we have studied their respective regulatory roles in detail by deletion and mutation analyses. We propose here, that the parasite is having an effect on the upregulation of the MMP9 gene via the induction of such transcription factors.

Life (proliferation) versus death (apoptosis) signals in Theileria-transformed lymphocytes

Gordon Langsley. Institut Pasteur, Paris.

Martin Baumgartner, Marie Chaussepied, Alphonse Garcia, Marie-Françoise Moreau and Gordon Langsley. Institut Pasteur, Paris. Theileria parasites modulate Src-family kinases and PI3-Kinase to control activation of AP-1 and proliferation in B-cells.

Bovine leukocytes infected by Theileria parasites behave as transformed cells capable of infiltrating different host lymphoid and non lymphoid tissues. Leukocyte proliferation and metastasis have been linked to the constitutive induction of the transcription factor AP-1. We have established that AP-1 transcriptional activity depends exclusively on JNK kinase as its dephosphorylation leads to AP-1 inhibition. Here, we report the contribution of Src kinases and PI3-K to both AP-1 induction and proliferation of Theileria-transformed leukocytes. Pharmacological inhibition of Src kinases by PP1 leads to down regulation of AP-1 activity and leukocyte growth arrest. Consistently, over expression of the negative Src-regulator C-terminal Src kinase (Csk) also reduced AP-1 activity. The Src kinase Hck is constitutively active in infected cells and modulated by the parasite, whereas Lyn, the major B cell Src kinase, is not involved. Surprisingly, elimination of the parasite from the host cell cytoplasma leads to activation of Hck over constitutive levels. We are currently analysing proteins that interact with Hck in a parasite-dependent manner and potentially regulate and/or target its kinase activity.

The lipid phosphatidylinositol 3-kinase (PI3-K) is constitutively activated in Theileria-infected B cells and its activity depends on the presence of the parasite within the host cell cytoplasm. PI3-K is involved in signalling to AP-1 and its pharmacological inhibition leads to down regulation of AP-1 activity and growth arrest, without inducing apoptosis. We are currently examining whether PKB/Akt and E2F, two known downstream targets of PI3-K, are activated and involved in the continuous proliferation of infected B cells. Moreover, we will address the question whether Src kinases and PI3-K signal by the same or separate pathways to activate AP-1.

Theileria-dependent activation of survival signals in infected and immortalised B-lymphocytes

M. Chaussepied, M-F. Moreau, D. Ginsberg* and G. Langsley. Department of Immunology, Pasteur Institute, Paris.* Department of Molecular and Cell Biology, Weizmann Institute of Science, Israel.

Theileria parasites infect bovine lymphocytes and induce both continuous proliferation and metastasis, reviewed in [1]. Since the intra-cellular parasite and the host cell divide synchronously each daugter lymphocyte remains infected and therfore, immortalised and can be cultured in vitro without the addition of any specific growth factor. Nonetheless, theilericidal treatment with buparvaquone (BW720c) reverts the lymphoblastoma phenotype and as the parasite dies the lymphocyte enters quiescence.

The 5-lipoxygenase inhibitor and free radical scavenging compound NDGA blocks proliferation of T. parva-infected B-cell line TpM409B2. Flow cytometric DNA content analysis of NDGA-treated TpM409B2 cells revealed several defects in cell cycle progression : slower transition from G1 to S, accumulation in G2-M and apoptosis of G1-S cells.

In TpM409B2 cells, as described for T. parva infected T-lymphocytes [2], NF-?B is activated. NDGA mediated unballance in redox cell status decreases Theileria-induced NF-?B activation. To address the potential role of NF-?B in promoting survival of Theileria-transformed B-lymphocytes, we performe cell cycle analysis on TpM409B2 cells transiently transfected with I-?Ba. Although transient overexpression of I-?Ba efficiently blocks NF-?B transactivating activity, transfected cells did not undergo programmed cell death, nor exhibit any alteration in mitotic cell cycle progression. However, it has been proposed that constitutive activation of NF-?B mediates an IL-2 dependent autocrine loop in Theileria infected T-lymphocytes [2]. Thus, in an I-?Ba transiently transfected B-cell population its is conceivable that the large percentage of non-transfected cells provide in trans, the growth factors/cytokines that could compensate for the loss of a NF-?B driven survival signal. We are constructing an inducible I-?Ba stable TpM409B2 cell line to assess the role of NF-?B activation in Theileria dependent immortalisation of B-lymphocytes. We are also examining the affect of NDGA treatment on Theileria-transformed B-lymphocyte cyclin expression. Preliminary experiments provide a tentative explanation for the observed G2-M block, since in NDGA-treated cells the G2-M population is cyclin B low, whereas cyclin A expression appears normal. Another NDGA target in TpM409B2 cells is the transcription factor E2F, a known mediator of S-phase entry and apoptosis [3]. We have observed the disappearence of specific E2F complexes upon NDGA treament. Experiments are underway to determine if E2F inhibition, via overexpression of mutant DP1 and DP2 (E2F dimerizing partners) mimics NDGA-induced failure in cell cycle progression and apoptosis of G1-S cells.

  1. M. Chausspied and G. Langsley (1996). Res. Immunol. 147, 127.
  2. V.T. Heussler et al. (1992). J. Immunol. 149, 562.
  3. Qin et al. (1994) PNAS 91, 10918-10922.

Theileria parva survives by inducing survival

Dirk Dobbelaere, Volker Heussler, Paula Fernandez and Joel Machado Jr. Inst. of Animal Pathology, Department of Molecular Pathology, Berne, Switzerland.

The intracellular parasite Theileria parva infects and transforms T-cells by activating a program of gene expression that enables the parasitised cell to proliferate in an uncontrolled manner, independent of antigenic stimulation and exogenous growth factors. The transcription factor NF-?B which regulates a number of genes that are involved in the activation and proliferation of T-cells is permanently activated in T. parva-transformed T-cells. This involves the constitutive degradation of two cytoplasmic NF-?B inhibitor proteins, I?Ba and I?Bß. In addition to contributing to proliferation, NF-?B has also been shown to protect cells against apoptosis induced by a range of stimuli, including tumour necrosis factor and chemotherapeutic agents. We examined the biological significance of constitutive NF-?B activity in T. parva-transformed T-cells. Blocking the phosphorylation and degradation of I?B? results in the abrupt induction of apoptosis in parasitised cells. Apoptosis correlates closely with reduced NF-?B DNA binding and transcriptional activity. In addition, interference with members of the NF-?B activation pathway by the expression of dominant negative mutant forms of NF-?B and I?B results in rapid apoptosis. These data demonstrate that permanent parasite-dependent activation of NF-?B blocks a constitutive apoptotic signal and is therefore an essential component in the transformation process.

Theileria DNA binding proteins and their interaction with the host - A family affair?

David G. Swan, Kirsten Philips, Rowena Stern, Laura Stadler, Sue McKellar, Andy Tait and Brian R. Shiels. Dept. of Veterinary Parasitology, University of Glasgow.

Theileria annulata is an apicomplexan protozoan parasite of Bovines. Infection of bovine lymphocytes by T. annulata induces the proliferation of the host cell. Proliferation is dependent on the presence of the parasite, as treatment with an anti-theileriacidal drug, buparvaquone, causes cell death. We are studying the molecular changes which occur as the parasite differentiates from the intracellular multinucleate stage to the extracellular merozoite stage (merogony). During merogony, the rate of host cell proliferation decrease and then ceases. It has previously been proposed that the parasite factor(s) which control infected-cell proliferation will be switched off during merogony. In this communication we describe the analysis of a small family of homologous genes, (TashAT), clustered in the genome of T. annulata whose expression is switched off relatively early during merogony. We present evidence that both TashAT1 and TashAT2 bear a domain containing the AT hook DNA-binding motif and that the AT hook domain from TashAT2 can bind with some specificity to AT rich DNA. Immunolocalisation, combined with experiments using inhibitors of protein translation, demonstrate that a likely location for TashAT2 is the host cell nucleus. That TashAT2 does encode the information required for transport to the nuclei of mammalian cells was provided by transfection experiments.

Is the virulence of Theileria annulata linked to three parasite antigens which interfere with antigen-specific T cell responses?

Patricia M. Preston. Institute of Cell, Animal and Population Biology, University of Edinburgh.

It is now generally accepted that Theileria annulata inhabits macrophages and that the cytokines produced by the infected cells may not only trigger innate immune mechanisms and help present antigen to CD4+ T cells but also, if the infected macrophages are present in significant numbers, induce the characteristic clinical symptoms of disease. Recent work on two schizont antigens and a third molecule produced by schizont-infected cells has provided additional reasons why cattle infected with high doses of parasites fail to control large populations of parasitised cells. Two schizont antigens, which are also expressed on the surface of infected host cells, appear to be the means by which schizont-infected cells stimulate lymphocytes to proliferate non-specifically in vitro. This phenomenon is believed to occur in cattle and prevent the induction of antigen-specific T cell responses. The third molecule produced by the infected cells suppresses interleukin-2-induced proliferation of lymphocytes in vitro raising the question as to whether paracortical T cells fail to proliferate in the lymph nodes of cattle undergoing lethal diseases because the parasitised cells are producing this factor. Such effective interference in the host's capacity to mount antigen-specific T cell responses would seem a good reason for proposing these three molecules as candidate virulence factors of T. annulata schizont-infected cells. Finding that the expression of one of the antigens, at least, was lost during attenuation of a virulent cell line supports this contention.

The limits for eukaryotic life

Dave Roberts. Natural History Museum.

The microbiology of extreme environments is usually considered the preserve of the Archaea and a few exotic Bacteria. In fact most extreme environments also contain microbial eukaryotes when there is sufficient food present to support them. Considered overall, eukaryotes living in extreme environments do not look very different from their mesophilic relatives, suggesting that the evolution of mechanisms to deal with extreme conditions has evolved many times in many groups. Thanks to their compartmentalised architecture, eukaryotes are capable of thriving in lower pH than any prokaryote for example. Only at high-temperatures do the prokaryotes have the field to themselves, as far as is presently known.

Microbiology of the deep-sea benthic boundary layer

C.M. Turley and J. Dixon. Plymouth Marine Laboratory. .

The deep-sea represents a significant long-term sink in the global carbon budget and can effectively remove carbon for hundreds (deep waters) to millions of years (geological sediment). Bacteria in deep-sea sediments constitute the largest global fraction of total benthic bacteria.

In the deep-sea benthic boundary layer, the dynamic interface between the sediment and the water column, the temperatures are low, pressures high and food is severely limiting. Particulate organic matter raining from the richer productive surface layer of the ocean, in the form of aggregates ("marine snow"), is the nutritional basis for life. "Marine snow" are sites of enrichment and support mostly small bacterivorous flagellates during their descent to the sea bed. Different species may have different pressure tolerances.

Respiration is dominated by bacteria and they play a major role in the decomposition of material on the deep-sea bed. Many other taxa, including Protista, respond to such seasonal influx of material such that populations of opportunist species may increase. Bacterivorous, barophillic, flagellates (Protozoa, Bodoninae), are commonly found by enrichment of water directly overlying the sediment or the sediment pore water. Under 450 atm and 2°C, Bodo sp. doubled every 2.11 days and had a growth efficiency of 17-25%. The step in the microbial food web from bacteria to flagellate could be important for remineralization and decomposition of sedimented biogenic particles and energy flow in the deep sea.

Cryptosporidium: surviving outside and inside the host

V. McDonald. St Bartholomew's and Royal London School of Medicine and Dentistry, London.

Cryptosporidium parvum is a coccidian which develops in mucosal cells of the gastrointestinal tract and causes a debilitating cholera-like diarrhoea. Infection is transmitted in a faecal-oral manner by oocysts which are commonly found in aquatic systems and contaminate public drinking water supplies. The oocyst comprises four sporozoites enclosed within a spherical proteinaceous shell which is highly resistant to environmental stress. Studies suggest that the cyst form may survive outside the host for many months and is resistant to commonly used sterilisation and water purification procedures. The signals for excystation of the sporozoites are poorly understood, but factors including temperature, acidity and the presence of intestinal contents may be important. The excysted sporozoites are able to survive the hostile environment of the intestinal lumen and can actively penetrate mucus to arrive at the host cell. The intracellular parasite occupies a unique extracytoplasmic location at the lumenal surface, but the significance of this developmental feature is unknown. Following invasion numerous parasite antigens are expressed on the surface of the infected cell and this alteration of the host cell membrane probably plays an important role in the intracellular growth of the parasite.

Survival strategies in lacustrine polar protozoa

Johanna Laybourn-Parry, Elanor Bell and Emily Roberts. University of Nottingham.

Contrary to what one might expect Antarctic lake protozooplankton do not shut-up shop for the winter, but maintain active populations, using various adaptations to achieve this. For example, coloured flagellates may resort to pure heterotrophy. Preliminary work on Arctic lakes indicates that population numbers drop to zero during the winter and encystment is probably the means of survival. The different survival strategies and the mechanisms by which they are achieved, will be discussed with reference to data from lakes in the Vestfold Hills, eastern Antarctica, the McMurdo Dry Valleys, western Antarctic and Svalbard (Arctic).

Strategies for microbial success in the Arctic and Antarctic

Warwick Vincent and Connie Lovejoy. Laval University, Québec, Canada.

Research in the north and south polar regions over the last decade has underscored the remarkable diversity of extreme habitats for protozoa and other microbiota. These high latitude communities face a variety of challenges: persistent low temperatures, disruptive effects of freeze-thaw cycles, extreme seasonality of energy supply, and pronounced variations in other properties of their environment. Despite these extremes, the microbiota achieve high local biomass levels and a surprisingly rich species diversity. These oases of microbial richness (for example, in meltpools on the Ward Hunt Ice Shelf, and in the open waters of polynyas) point to strategies to maximize energy capture during the brief growing season while minimizing loss rates, for example during winter darkness. The microbiota of the polar regions must now contend with additional stresses associated with anthropogenic impacts; in particular, the dispersal and concentration of pollutants, stratospheric ozone depletion, and climate change resulting from increased greenhouse gases. These effects are operating at a global scale, but are amplified in the polar regions. Our work indicates that polar microbial communities are well equipped to adjust to these perturbations but in the process they are likely to shift in species composition and size structure. Some protozoa appear to profit from increases in UV and temperature, and these taxa may play an increasing role in polar aquatic food webs.