Malaria Programme: Rayner group

The Malaria Programme uses genomic and genetic approaches to discover molecular mechanisms of host-parasite interactions that may lead to new biological insights and improved strategies for disease prevention.

Plasmodium falciparum is the parasite responsible for the overwhelming majority of malaria mortality.

The Rayner group is investigating the molecular details of the interactions between P. falciparum and human red blood cells (erythrocytes), using both genomic and proteomic approaches.

The Rayner lab has a particular focus on erythrocyte invasion, the process by which P. falciparum parasites recognise and invade human red blood cells. This is a critical step in the parasite's life cycle, because once inside erythrocytes, P. falciparum parasites are protected from the antibody-mediated immune system, and use the hemoglobin within the blood cell to grow and multiply. By understanding erythrocyte invasion in depth, the Rayner group aims to identify molecular steps in the process that could be targeted by new drugs or vaccines.

More information on the Malaria Programme.

[David Goulding, Genome Research Limited]

Background

Figure 1. Erythrocyte invasion.

Figure 1. Erythrocyte invasion.

Erythrocyte invasion: a complex dance between two genomes

All the symptoms and pathology of malaria are initiated when the invasive stage of P. falciparum, called merozoites, infect erythrocytes, leading to the colonisation and eventual destruction of these erythrocytes and the release of a new wave of invasive merozoites 48 hours later. Erythrocyte invasion is critical for parasite survival because P. falciparum is an intracellular parasite - the merozoite can not exist outside of an erythrocyte for any length of time. It is also a complex point of interaction between two genomes and the proteins that they encode.

Erythrocyte invasion consists of several phases. Recognition and attachment of merozoites to erythrocytes is driven by multiple interactions between proteins present on the surface of the merozoite and it's target erythrocyte. Although many P. falciparum ligands thought to play a role in this process have been identified, in most cases it is not known what erythrocyte receptors they bind to, or what their precise function is. This initial interaction step is further complicated by genetic variation in both the host and parasite genomes, which can dictate which erythrocytes the P. falciparum merozoites can recognise. Understanding the receptor-ligand interactions that mediate merozoite-erythrocyte attachment, and how those interactions are influenced by natural genetic variation, is one of the main goals of the Rayner group.

Once attachment has occurred, the P. falciparum merozoite literally drives its way into the erythrocyte, a process that is entirely driven by an actin-myosin motor within the parasite, similar to the actin myosin motors that drive muscle contraction. This invasion motor is in turn regulated by a complex web of post-translational modifications, and understanding these regulatory networks is another key goal of the Rayner group.

Research

Figure 2. By labeling a sub-population of erythrocytes with a fluorescent dye (green), and parasites with a fluorescent dye of a different colour (blue), we can use flow cytometry to count how many parasites have invaded those erythrocytes.

Figure 2. By labeling a sub-population of erythrocytes with a fluorescent dye (green), and parasites with a fluorescent dye of a different colour (blue), we can use flow cytometry to count how many parasites have invaded those erythrocytes.

zoom

The Rayner group aims to develop a better understanding of the molecular details of host-parasite interactions during the P. falciparum blood stages, with a particular focus on erythrocyte invasion. To achieve our aims we use a variety of approaches, many of which make use of the unique strengths of the Sanger Institute. Tiny changes in the DNA sequence called single nucleotide polymorphisms (SNPs) are known to be responsible for a lot of the variation seen in the DNA sequences of organisms. Genome-wide studies in the Sanger Institute's Malaria Programme are now identifying numerous SNPs within both human erythrocyte receptors and P. falciparum invasion ligands (Malaria Programme: Kwiatkowski group), but their effect on receptor-ligand binding or invasion is not yet known. We are particularly interested in developing new high-throughput phenotyping platforms to assess the effect that these SNPs have on specific steps during erythrocyte invasion, and have recently developed a two-colour flow cytometry based assay to quantitate erythrocyte invasion.

We are also using experimental genetic approaches to dissect the molecular roles of specific P. falciparum proteins in the process of erythrocyte invasion. We work with Gavin Wright's team to identify new receptor-ligand interactions involved in invasion, and to measure the impact of natural genetic variation on these interactions. In collaboration with Oliver Billker and the Sanger Institute Proteomics Mass Spectrometry core facility led by Jyoti Choudhary we are using mass spectrometry to study two posttranslational modifications that play key roles in regulating erythrocyte invasion - phosphorylation and palmitoylation.

Finally, we play a leading role in a global collaboration aimed at understanding the origins of P. falciparum parasites and what restricts certain parasites to certain hosts. In a recent comprehensive genetic study of parasites infecting wild apes in central Africa, we established that Plasmodium infection is widespread among apes, with at least six apparently independent P. falciparum related species present. All human P. falciparum parasites fall within a single clade of parasites infecting western lowland gorillas, arguing that Plasmodium falciparum passed from gorillas to humans at some time during our evolutionary history. Further studies are aimed at understanding whether such cross-species transmissions still occur and what the molecular requirements are that control which host species a Plasmodium parasite can infect.

Resources

Standard operating procedure for Plasmodium falciparum erythrocyte invasion assay - This document contains a detailed SOP for the two-colour flow cytometric erythrocyte invasion assay recently developed by the Rayner lab (Theron et al., Cytometry Part A, in press). It contains step by step methods for labeling erythrocytes using CFDA SE or DDAO SE, labeling parasite DNA with Hoechst 33342 or SYBR Green I, and how to combine erythrocyte and DNA dyes to measure P. falciparum invasion.

For further information, contact Leyla Bustamante.

Selected Publications

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Nature 2011;480;7378;534-7

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    Nature methods 2011;8;12;1078-82

  • A plethora of Plasmodium species in wild apes: a source of human infection?

    Rayner JC, Liu W, Peeters M, Sharp PM and Hahn BH

    Trends in parasitology 2011;27;5;222-9

  • Malaria immunoepidemiology in low transmission: correlation of infecting genotype and immune response to domains of Plasmodium falciparum merozoite surface protein 3.

    Jordan SJ, Oliveira AL, Hernandez JN, Oster RA, Chattopadhyay D, Branch OH and Rayner JC

    Infection and immunity 2011;79;5;2070-8

  • An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

    Theron M, Hesketh RL, Subramanian S and Rayner JC

    Cytometry. Part A : the journal of the International Society for Analytical Cytology 2010;77;11;1067-74

  • Origin of the human malaria parasite Plasmodium falciparum in gorillas.

    Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, Keele BF, Ndjango JB, Sanz CM, Morgan DB, Locatelli S, Gonder MK, Kranzusch PJ, Walsh PD, Delaporte E, Mpoudi-Ngole E, Georgiev AV, Muller MN, Shaw GM, Peeters M, Sharp PM, Rayner JC and Hahn BH

    Nature 2010;467;7314;420-5

  • Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex.

    Jones ML, Kitson EL and Rayner JC

    Molecular and biochemical parasitology 2006;147;1;74-84

The Team

Team members

Leyla Bustamante Rodriguez
lbr@sanger.ac.ukScientific Manager
Matthew Jones
mj5@sanger.ac.ukunknown
Alena Pance
Staff Scientist
Will Proto
Postdoctoral Fellow
Jennifer Volz
unknown

Leyla Bustamante Rodriguez

lbr@sanger.ac.uk Scientific Manager

I completed my first degree at the Universidad Nacional in Bogota, Colombia, where I undertook a project examining the release of Plasmodium falciparum merozoites from erythrocytes. I then worked at CIDEIM in Cali, Colombia, researching molecular markers of drug resistant malaria, and in 2005 I completed my PhD at the Universidad Complutense in Spain, investigating the expression of the oxidative cascade genes in P. falciparum. I then moved to St. George's, University of London, in the UK as a postdoctoral fellow studying mechanisms of action and resistance to artemisinins in P. falciparum. I joined the Sanger Institute in 2009.

Research

I am currently the Scientific Manager for Julian Rayner's team and my projects include invasion phenotyping assays and the study of interactions between Plasmodium proteins and receptors on the erythrocyte surface.

References

  • Biochemical and functional analysis of two Plasmodium falciparum blood-stage 6-cys proteins: P12 and P41.

    Taechalertpaisarn T, Crosnier C, Bartholdson SJ, Hodder AN, Thompson J, Bustamante LY, Wilson DW, Sanders PR, Wright GJ, Rayner JC, Cowman AF, Gilson PR and Crabb BS

    Burnet Institute, Melbourne, Victoria, Australia.

    The genomes of Plasmodium parasites that cause malaria in humans, other primates, birds, and rodents all encode multiple 6-cys proteins. Distinct 6-cys protein family members reside on the surface at each extracellular life cycle stage and those on the surface of liver infective and sexual stages have been shown to play important roles in hepatocyte growth and fertilization respectively. However, 6-cys proteins associated with the blood-stage forms of the parasite have no known function. Here we investigate the biochemical nature and function of two blood-stage 6-cys proteins in Plasmodium falciparum, the most pathogenic species to afflict humans. We show that native P12 and P41 form a stable heterodimer on the infective merozoite surface and are secreted following invasion, but could find no evidence that this complex mediates erythrocyte-receptor binding. That P12 and P41 do not appear to have a major role as adhesins to erythrocyte receptors was supported by the observation that antisera to these proteins did not substantially inhibit erythrocyte invasion. To investigate other functional roles for these proteins their genes were successfully disrupted in P. falciparum, however P12 and P41 knockout parasites grew at normal rates in vitro and displayed no other obvious phenotypic changes. It now appears likely that these blood-stage 6-cys proteins operate as a pair and play redundant roles either in erythrocyte invasion or in host-immune interactions.

    Funded by: Wellcome Trust: 077108/Z/05/Z

    PloS one 2012;7;7;e41937

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

    Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

    Funded by: Medical Research Council: G19/9; NCEZID CDC HHS: R36 CK000119-01; NIAID NIH HHS: 2T32 AI007535-12, R01 AI057919, R01 AI057919-05, R01AI057919; Wellcome Trust: 077108, 089084, 090532

    Nature 2011;480;7378;534-7

  • Mechanisms of artemisinin action and resistance: wider focus is needed.

    Woodrow CJ and Bustamante LY

    Trends in parasitology 2011;27;1;2-3; author reply 3-4

  • Plasmodium falciparum ATP6 not under selection during introduction of artemisinin combination therapy in Peru.

    Woodrow CJ and Bustamante LY

    Antimicrobial agents and chemotherapy 2010;54;5;2280; author reply 2280-1

  • Effect of artemisinins and amino alcohol partner antimalarials on mammalian sarcoendoplasmic reticulum calcium adenosine triphosphatase activity.

    Toovey S, Bustamante LY, Uhlemann AC, East JM and Krishna S

    Academic Centre for Travel Medicine & Vaccines, WHO Collaborating Centre for Travel Medicine, Royal Free & University College Medical School, London, UK. malaria@freesurf.ch

    The aim of this study was to assess the ability of currently deployed antimalarials to inhibit mammalian sarcoendoplasmic reticulum calcium adenosine triphosphatase (SERCA). Artemisinins exert their antiplasmodial action by inhibiting parasite PfATP6, a SERCA enzyme, and possess neurotoxic potential; mefloquine is neurotoxic and inhibits mammalian SERCA, an orthologue of PfATP6. SERCA in rabbit muscle was tested in vitro for inhibition by artemisinin and amino alcohol antimalarials. Significant inhibition of mammalian SERCA, as mean difference from uninhibited, control values was seen with both enantiomers of mefloquine: (+)-mefloquine (10 microM: -35.83, 95% CI -59.63 to -12.03; 50 microM: -54.06, 95% CI -77.86 to -30.26); (-)-mefloquine (10 microM: -24.35, 95% CI -41.56 to -7.15; 50 microM: -58.42, 95% CI -75.62 to -41.22); lumefantrine (1 microM: -25.46, 95% CI -45.82 to -5.10; 5 microM -34.83, 95% CI -60.08 to -9.58; 10 microM: -25.80, 95% CI -51.05 to -0.55); desbutyl-lumefantrine (5 microM: -50.16, 95% CI -84.24 to -16.08); dihydroartemisinin (1 microM: -39.25, 95% CI -63.74 to -14.76; 5 microM: -39.30, 95% CI -64.88 to -13.72). Dihydroartemisinin in higher concentrations (10 microM) stimulated SERCA activity: (+40.90, 95% CI 11.37 to 70.44). No statistically significant inhibition was seen with artemether at 1, 5 and 10 microM. Equimolar combinations of artemether and lumefantrine or of dihydroartemisinin and lumefantrine, when studied at concentrations that inhibit SERCA individually, failed to show any inhibition. Dihydroartemisinin, mefloquine, lumefantrine and desbutyl lumefantrine inhibit mammalian SERCA at periphysiological concentrations, although the neurotoxicity of mefloquine is not wholly attributable to this property. Candidate antimalarials should be screened pre-clinically for SERCA inhibition.

    Funded by: Wellcome Trust: 074395/Z/04/Z

    Basic & clinical pharmacology & toxicology 2008;103;3;209-13

  • The role of pfmdr1 in Plasmodium falciparum tolerance to artemether-lumefantrine in Africa.

    Sisowath C, Ferreira PE, Bustamante LY, Dahlström S, Mårtensson A, Björkman A, Krishna S and Gil JP

    Malaria Research Unit, Division of Infectious Diseases, Department of Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden. christin.sisowath@ki.se

    Objective: Artemether-lumefantrine (AL), presently the most favoured combination therapy against uncomplicated Plasmodium falciparum malaria in Africa, has recently shown to select for the pfmdr1 86N allele. The objective of this study was to search for the selection of other mutations potentially involved in artemether-lumefantrine tolerance and/or resistance, i.e. pfmdr1 gene amplification, pfmdr1 Y184F, S1034C, N1042D, D1246Y, pfcrt S163R and PfATP6 S769N.

    Methods: The above mentioned SNPs were analysed by PCR-restriction fragment length polymorphism and pfmdr1 gene amplification by real-time PCR based protocols in parasites from 200 children treated with AL for uncomplicated P. falciparum malaria in Zanzibar.

    Results: A statistically significant selection of pfmdr1 184F mostly in combination with 86N was seen in reinfections after treatment. No pfmdr1 gene amplification was found.

    Conclusion: The results suggest that different pfmdr1 alleles are involved in the development of tolerance/resistance to lumefantrine.

    Tropical medicine & international health : TM & IH 2007;12;6;736-42

  • Mechanism of antimalarial action of the synthetic trioxolane RBX11160 (OZ277).

    Uhlemann AC, Wittlin S, Matile H, Bustamante LY and Krishna S

    Division of Cellular and Molecular Medicine, Centre for Infection, St. George's University of London, and Dept. of Infectious Diseases, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom.

    RBX11160 (OZ277) is a fully synthetic peroxidic antimalarial in clinical development. To study the possible mechanisms of action of RBX11160, we have examined its ability to inhibit PfATP6, a sarcoplasmic reticulum calcium ATPase and proposed target for semisynthetic peroxidic artemisinin derivatives. RBX11160 inhibits PfATP6 (apparent half-maximal inhibitory constant=7,700 nM) less potently than artemisinin (79 nM). Inhibition of PfATP6 is abrogated by desferrioxamine, an iron-chelating agent. Consistent with this finding, the killing of Plasmodium falciparum organisms by RBX11160 in vitro is antagonized by desferrioxamine. Artesunate and RBX11160 also act antagonistically against P. falciparum in vitro. A fluorescent derivative of RBX11160 localizes to the parasite cytosol in some parasites and to the food vacuole in other parasites. These data demonstrate that there are both similarities and differences between the antimalarial properties of RBX11160 and those of semisynthetic antimalarials such as artesunate and artemisinin.

    Antimicrobial agents and chemotherapy 2007;51;2;667-72

  • Dual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum.

    Bustamante LY, Crooke A, Martínez J, Díez A and Bautista JM

    Universidad Complutense de Madrid, Madrid, Spain.

    The genome of the human malaria parasite Plasmodium falciparum is extremely AT-rich such that it is particularly difficult to design standard probes to identify and quantify specific transcripts. Biased AT genome contents (70%-80%) lead to a high proportion of short repetitions and a low free energy of binding between target sequences and their specific probes during hybridization. This causes nonspecific annealing and high background noise. We constructed molecular beacon probes with dual-function stems to avoid nonspecific detection and establish identical melting patterns for use with several fluorescent probes for the analysis of mRNA expression in P. falciparum in real-time reverse transcription PCR (RT-PCR) assays. The method proved highly efficient at detecting low transcript levels in P. falciparum microcultures. Conditions were established for two types of real-time instruments, demonstrating that molecular beacons with dual-function stems are a useful tool for the functional analysis of high AT genomes. The procedure could be adapted to high-throughput gene expression protocols for the biomolecular screening of the P. falciparum and other AT-rich genomes.

    BioTechniques 2004;36;3;488-92, 494

  • Release of merozoites from Plasmodium falciparum-infected erythrocytes could be mediated by a non-explosive event.

    Winograd E, Clavijo CA, Bustamante LY and Jaramillo M

    Laboratorio de Biologia Celullar, Instituto Nacional de Salud, Bogota, Colombia. enwino@hotmail.com

    Little is known about the molecular mechanism underlying the release of merozoites from malaria-infected erythrocytes. In the present study, video microscopy was carried out, and images throughout the process of merozoite release from Plasmodium falciparum-infected erythrocytes were digitized and analyzed. Merozoites were shown to escape from the infected host cell in about 1 s through a single site of the infected erythrocyte membrane, whose dimension was estimated to be 2.5 microm. Merozoites were released together with the residual body containing hemozoin, leaving behind a membranous structure that persisted even after an extended period of observation. Densitometric measurements showed that the cytoplasmic content of the infected erythrocyte did not diffuse out as parasites were released, but was gradually lost thereafter. This would indicate that the release of merozoites from infected erythrocytes is not mediated by an explosive event.

    Parasitology research 1999;85;8-9;621-4

Matthew Jones

mj5@sanger.ac.uk unknown

I completed my undergraduate work at Shorter College, a small school in the Southeastern U.S. After completion of my undergraduate training, I moved to the University of Alabama at Birmingham and took up a PhD project centering on the mechanisms underlying red blood cell invasion by the malaria-causing parasite, Plasmodium falciparum. This work was completed in the laboratory of Julian Rayner, and upon completing my PhD work, I moved with Dr. Rayner to the Sanger Institute to continue working with P. falciparum.

Research

I currently am involved in the characterization of specific post translation modifications that affect P. falciparum blood-stage development.

References

  • Effects of calcium signaling on Plasmodium falciparum erythrocyte invasion and post-translational modification of gliding-associated protein 45 (PfGAP45).

    Jones ML, Cottingham C and Rayner JC

    Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

    Plasmodium falciparum erythrocyte invasion is powered by an actin/myosin motor complex that is linked both to the tight junction and to the merozoite cytoskeleton through the Inner Membrane Complex (IMC). The IMC association of the myosin motor, PfMyoA, is maintained by its association with three proteins: PfMTIP, a myosin light chain, PfGAP45, an IMC peripheral membrane protein, and PfGAP50, an integral membrane protein of the IMC. This protein complex is referred to as the glideosome, and given its central role in erythrocyte invasion, this complex is likely the target of several specific regulatory effectors that ensure it is properly localized, assembled, and activated as the merozoite prepares to invade its target cell. However, little is known about how erythrocyte invasion as a whole is regulated, or about how or whether that regulation impacts the glideosome. Here we show that P. falciparum erythrocyte invasion is regulated by the release of intracellular calcium via the cyclic-ADP Ribose (cADPR) pathway, but that inhibition of cADPR-mediated calcium release does not affect PfGAP45 phosphorylation or glideosome association. By contrast, the serine/threonine kinase inhibitor, staurosporine, affects both PfGAP45 isoform distribution and the integrity of the glideosome complex. This data identifies specific regulatory elements involved in controlling P. falciparum erythrocyte invasion and reveals that the assembly status of the merozoite glideosome, which is central to erythrocyte invasion, is surprisingly dynamic.

    Funded by: NIAID NIH HHS: T32 AI055438, T32 AI055438-05

    Molecular and biochemical parasitology 2009;168;1;55-62

Alena Pance

- Staff Scientist

After completing a first degree in biology and a masters degree on cell biology and tropical medicine in Venezuela, Alena Pance moved to England to do a PhD in the Department of Biochemistry at the University of Cambridge. The first post-doctoral position was in the Faculty of Medicine, Univeristy of Burgundy in Dijon France, followed by an Individual Marie Curie Research Fellowship to come back to Biochemistry, Cambridge.

Research

The research at the Sanger Insitute focuses on the mechanisms of erythrocyte invasion by P. falciparum. The approach for these studies is the use of human IPS cells differentiated into erythrocytes, which allows us to genetically manipulate the host cell to explore the role of surface receptors implicated in parasite invasion. This fits into examining the host's side of the host-parasite interaction, which is the main goal of the group.

References

  • A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory-deficient PC12 cells.

    Pance A, Livesey FJ and Jackson AP

    Department of Biochemistry, University of Cambridge, Cambridge, UK.

    The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down-regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1-silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant-negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the beta3 subunit of the voltage-gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re-expressed. Granule and synaptic vesicle proteins were not re-expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post-transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory-deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

    Journal of neurochemistry 2006;99;5;1435-44

  • Nitric oxide and hormones in breast cancer: allies or enemies?

    Pance A

    Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK. a.pance@bioc.cam.ac.uk

    Unlike other types of cancer, tumors of the breast are greatly influenced by steroid hormones. The effect of estrogen and progesterone depends on the presence of their specific receptors and these constitute important parameters in determining the aggressiveness of the tumor, the feasibility of certain therapies and the prediction of relapse. The molecular mechanisms of steroid hormone action have not been fully elucidated but recent findings implicate the nitric oxide (NO) pathway in some of these effects. Both hormones can regulate the nitric oxide synthases (NOS) and, in turn, the NO produced has profound consequences on tumor cell homeostasis. On one hand, estrogen increases the activity of endothelial NOS (eNOS or NOSIII), while progesterone activates inducible NOS (iNOS or NOSII) expression. The data presented suggest that the low levels of NO produced by NOSIII mediate the proliferative effect of estrogen. On the other hand, the increase in apoptosis in response to progesterone could implicate the high levels of NO produced by induction of NOSII expression. Understanding of the mechanisms and interactions of steroid hormones with the NO pathway could lead to the development of new approaches and strategies for the effective treatment of breast cancer.

    Future oncology (London, England) 2006;2;2;275-88

  • Progesterone enhances cytokine-stimulated nitric oxide synthase II expression and cell death in human breast cancer cells.

    Bentrari F, Arnould L, Jackson AP, Jeannin JF and Pance A

    EPHE INSERM 517, Faculty of Medicine, University of Bourgogne, Dijon, France.

    The presence of hormone receptors is related to survival outcome in breast cancer. Previous results from our laboratory established a correlation between the presence of nitric oxide synthase II (NOSII) and nitric oxide (NO) production with progesterone receptors in a series of human breast tumours. Furthermore, this was directly related to a lower tumour grade and a lower proliferation rate of the tumour cells. To examine these results in further detail, the effect of progesterone (Pg) and 17beta-oestradiol (E2) on NOSII expression was analysed in the human breast cancer cell line MCF-7. By Northern blot and promoter activity, we show that a cytokine mix (TNF-alpha, IL-beta, and IFN-gamma) induces NOSII transcription after 6 h stimulation. In the absence of cytokines, neither hormone affects NOSII expression. However, Pg but not E2, enhances cytokine-induced NOSII transcription as well as NO synthesis, mainly by cooperation with gamma-interferon. The increase in NO accumulation in the media induced by addition of Pg to the cytokine treatment significantly increases cell death, mainly accounted for by apoptosis, as compared to the effect of cytokines alone. Our findings help clarify the role of steroid hormones in NOSII expression as well as the effect on cell viability and may suggest novel approaches towards hormonotherapy and the treatment of cancer.

    Laboratory investigation; a journal of technical methods and pathology 2005;85;5;624-32

  • Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant.

    Schmitt E, Parcellier A, Gurbuxani S, Cande C, Hammann A, Morales MC, Hunt CR, Dix DJ, Kroemer RT, Giordanetto F, Jäättelä M, Penninger JM, Pance A, Kroemer G and Garrido C

    Institut National de la Santé et de la Recherché Médicale U-517, Faculty of Medicine and Pharmacy, Dijon, France.

    Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.

    Cancer research 2003;63;23;8233-40

  • Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor.

    Gurbuxani S, Schmitt E, Cande C, Parcellier A, Hammann A, Daugas E, Kouranti I, Spahr C, Pance A, Kroemer G and Garrido C

    INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France.

    Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150-268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFDelta150-268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF-HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.

    Oncogene 2003;22;43;6669-78

  • A repressor in the proximal human inducible nitric oxide synthase promoter modulates transcriptional activation.

    Pance A, Chantome A, Reveneau S, Bentrari F and Jeannin JF

    Cancer Immunotherapy Laboratory of the Ecole Pratique des Hautes Etudes and INSERM U-517, University of Bourgogne, Faculty of Medicine, 21000 Dijon, France. apance@u-bourgogne.fr

    The human inducible nitric oxide synthase (iNOS or NOSII) gene is regulated through an extended and complex promoter. In this study, the transcriptional regulation of human NOSII is investigated in the human colon cell line HCT-8R. Stimulation with a cytokine mix (interferon-gamma, interleukin 1-beta, and tumor necrosis factor alpha) induces NOSII mRNA accumulation, as well as promoter activity in these cells. Several random deletions were performed within the proximal 7 kb of the promoter, which led to the identification of a region, whose deletion provokes a marked increase in transcriptional activity upon cytokine stimulation. Furthermore, this region is shown to repress a viral-driven luciferase construct, mainly at basal levels. An AP-1-like sequence present in this region that is specifically recognized by nuclear proteins is shown to be involved in the repressive effect. This element is capable of repressing a viral promoter, and its deletion augments cytokine-stimulated transcription. These findings are confirmed in various cell lines and suggest a general mechanism for the control of basal levels of NOSII expression, to avoid unnecessary toxicity under normal conditions.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002;16;6;631-3

  • Mechanisms of the antitumoral effect of lipid A.

    Reisser D, Pance A and Jeannin JF

    Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, 21079 Dijon, France. reisserd@u-bourgogne.fr

    Bacterial lipopolysaccharide (LPS) and its active component, lipid A, have been used either alone or as adjuvant in therapeutic anticancer vaccines. Lipid A induces various transcription factors via intracellular signaling cascades initiated by their receptor CD14-TLR4. These events lead to the synthesis of cytokines, which either have direct cytotoxic effect or stimulate the immune system. Their antitumoral effect has been demonstrated in animal models as well as clinical trials. Studies in animal models showed that their antitumoral effect relies mostly on the generation of an effective immune response. In humans, the antitumoral effect was correlated with an antibody response and cell-mediated cytotoxicity. So far, some encouraging results have been achieved in phase I and II clinical trials with regards to response and stabilization of the disease, but an expansion of the studies and trials is needed to find the best conditions for their clinical application.

    BioEssays : news and reviews in molecular, cellular and developmental biology 2002;24;3;284-9

  • Detection of specific antibodies to Plasmodium falciparum in blood bank donors from malaria-endemic and non-endemic areas of Venezuela.

    Contreras CE, Pance A, Marcano N, González N and Bianco N

    Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracas.

    Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

    The American journal of tropical medicine and hygiene 1999;60;6;948-53

Will Proto

- Postdoctoral Fellow

As an undergraduate I studied Microbiology at the University of Liverpool. In 2006, I moved to the University of Glasgow to undertake my PhD working in Jeremy Mottram’s lab. My PhD focused on investigating cysteine peptidases and autophagy in Trypanosoma brucei. After my PhD, I commenced a postdoctoral position in the same lab and expanded my research into cell death mechanisms of parasitic protozoa.

Research

In 2013, I joined Julian Rayner’s group at the Sanger Institute where I will study erythrocyte invasion by Plasmodium, investigating its potential influence on the host specificity of Plasmodium parasites.

References

  • Cell death in parasitic protozoa: regulated or incidental?

    Proto WR, Coombs GH and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

    Apoptosis and other types of regulated cell death have been defined as fundamental processes in plant and animal development, but the occurrence of, and possible roles for, regulated cell death in parasitic protozoa remain controversial. A key problem has been the difficulty in reconciling the presence of apparent morphological markers of apoptosis and the notable absence of some of the key executioners functioning in higher eukaryotes. Here, we review the evidence for regulated cell death pathways in selected parasitic protozoa and propose that cell death in these organisms be classified into just two primary types: necrosis and incidental death. It is our opinion that dedicated molecular machinery required for the initiation and execution of regulated cell death has yet to be convincingly identified.

    Funded by: Medical Research Council: 0700127, G9722968; Wellcome Trust: 085349

    Nature reviews. Microbiology 2013;11;1;58-66

  • Crystal structure of a Trypanosoma brucei metacaspase.

    McLuskey K, Rudolf J, Proto WR, Isaacs NW, Coombs GH, Moss CX and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom. karen.mcluskey@glasgow.ac.uk

    Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.

    Funded by: Medical Research Council: 0700127, G0700127; Wellcome Trust: 085349, 091790

    Proceedings of the National Academy of Sciences of the United States of America 2012;109;19;7469-74

  • Trypanosoma brucei metacaspase 4 is a pseudopeptidase and a virulence factor.

    Proto WR, Castanys-Munoz E, Black A, Tetley L, Moss CX, Juliano L, Coombs GH and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom.

    Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.

    Funded by: Medical Research Council: G0000508, G0700127, G9722968

    The Journal of biological chemistry 2011;286;46;39914-25

Jennifer Volz

- unknown

After graduating in Biology from Humboldt University, Berlin, Jennifer earned her PhD in 2004, researching the innate immune responses of Anopheles gambiae towards Plasmodium at the EMBL, Heidelberg, under the supervision of Prof Fotis Kafatos.

In 2005, Jennifer moved on to study Plasmodium falciparum blood stages in the laboratory of Prof Alan Cowman at the WEHI, Melbourne. During that time, she focussed on the identification and characterization of novel epigenetic regulators and their role in var gene expression.

Jennifer joined the Malaria Programme at the Wellcome Trust Sanger Institute in 2011.

Research

The Billker and Rayner labs have recently established a Plasmodium berghei pipeline, which allows the genetic modification of every gene in the parasite's genome one by one at scale. Jennifer's current role is to transfer this technology to Plasmodium falciparum.

References

  • PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division.

    Volz JC, Bártfai R, Petter M, Langer C, Josling GA, Tsuboi T, Schwach F, Baum J, Rayner JC, Stunnenberg HG, Duffy MF and Cowman AF

    The Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria, Australia.

    A major virulence factor of the malaria parasite Plasmodium falciparum is erythrocyte membrane protein 1 (PfEMP1), a variant protein expressed on the infected erythrocyte surface. PfEMP1 is responsible for adherence of infected erythrocytes to the endothelium and plays an important role in pathogenesis. Mutually exclusive transcription and switched expression of one of 60 var genes encoding PfEMP1 in each parasite genome provides a mechanism for antigenic variation. We report the identification of a parasite protein, designated PfSET10, which localizes exclusively to the perinuclear active var gene expression site. PfSET10 is a histone 3 lysine 4 methyltransferase required to maintain the active var gene in a poised state during division for reactivation in daughter parasites, and as such is required for P. falciparum antigenic variation. PfSET10 likely maintains the transcriptionally permissive chromatin environment of the active var promoter and thus retains memory for heritable transmission of epigenetic information during parasite division.

    Cell host & microbe 2012;11;1;7-18

  • A genome-wide chromatin-associated nuclear peroxiredoxin from the malaria parasite Plasmodium falciparum.

    Richard D, Bartfai R, Volz J, Ralph SA, Muller S, Stunnenberg HG and Cowman AF

    Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

    Malaria parasites are subjected to high levels of oxidative stress during their development inside erythrocytes and the ability of the parasite to defend itself against this assault is critical to its survival. Therefore, Plasmodium possesses an effective antioxidant defense system that could potentially be used as a target for the development of inhibitor-based therapy. We have identified an unusual peroxiredoxin protein that localizes to the nucleus of Plasmodium falciparum and have renamed it PfnPrx (PF10_0268, earlier called MCP1). Our work reveals that PfnPrx has a broad specificity of substrate being able to utilize thioredoxin and glutaredoxin as reductants and having the ability to reduce simple and complex peroxides. Intriguingly, chromatin immunoprecipitation followed by deep sequencing reveals that the enzyme associates with chromatin in a genome-wide manner with a slight enrichment in coding regions. Our results represent the first description of a dedicated chromatin-associated peroxiredoxin and potentially represent an ingenious way by which the parasite can survive the highly oxidative environment within its human host.

    Funded by: Canadian Institutes of Health Research

    The Journal of biological chemistry 2011;286;13;11746-55

  • Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter.

    Petter M, Lee CC, Byrne TJ, Boysen KE, Volz J, Ralph SA, Cowman AF, Brown GV and Duffy MF

    Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia.

    Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

    PLoS pathogens 2011;7;2;e1001292

  • Potential epigenetic regulatory proteins localise to distinct nuclear sub-compartments in Plasmodium falciparum.

    Volz J, Carvalho TG, Ralph SA, Gilson P, Thompson J, Tonkin CJ, Langer C, Crabb BS and Cowman AF

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia.

    The life cycle of the malaria parasite Plasmodium falciparum involves dramatic morphological and molecular changes required for infection of insect and mammalian hosts. Stage-specific gene expression is crucial, yet few nuclear factors, including potential epigenetic regulators, have been identified. Epigenetic mechanisms play an important role in the switched expression of members of species-specific gene families, which encode proteins exported into the cytoplasm and onto the surface of infected erythrocytes. This includes the large virulence-associated var gene family, in which monoallelic transcription of a single member and switching to other var genes leads to a display of different surface ligands with distinct antigenic and adhesive properties. Using a bio-informatic approach we identified 24 putative nuclear proteins. Tagging with sequences encoding GFP or haemagglutinin (HA) epitopes allowed for identification and localisation analysis of 12 nuclear proteins that are potential regulators of P. falciparum gene expression. These proteins specifically localise to distinct areas of the nucleus, reaching from the centre towards the nuclear envelope, giving new insights into the apicomplexan nuclear architecture. Proteins presenting a punctate distribution in the perinuclear sub-compartments are potential virulence gene regulators as silenced and active var genes reside at the nuclear periphery either clustered or in small expression sites, respectively. These analyses demonstrated an ordered compartmentalisation, indicating a complex sub-nuclear organisation that contributes to the complexity of transcriptional regulation in P. falciparum.

    Funded by: Howard Hughes Medical Institute

    International journal for parasitology 2010;40;1;109-21

  • Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors.

    Flueck C, Bartfai R, Volz J, Niederwieser I, Salcedo-Amaya AM, Alako BT, Ehlgen F, Ralph SA, Cowman AF, Bozdech Z, Stunnenberg HG and Voss TS

    Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, Basle, Switzerland.

    Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host-parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens.

    PLoS pathogens 2009;5;9;e1000569

  • A genetic module regulates the melanization response of Anopheles to Plasmodium.

    Volz J, Müller HM, Zdanowicz A, Kafatos FC and Osta MA

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.

    Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.

    Cellular microbiology 2006;8;9;1392-405

  • The roles of two clip domain serine proteases in innate immune responses of the malaria vector Anopheles gambiae.

    Volz J, Osta MA, Kafatos FC and Müller HM

    European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

    The malaria vector Anopheles gambiae is capable of multiple immune responses against Plasmodium ookinetes. Accumulating evidence in several insect species suggests the involvement of serine protease cascades in the initiation and coordination of immune responses. We report molecular and reverse genetic characterization of two mosquito clip domain serine proteases, CLIPB14 and CLIPB15, which share structural similarity to proteases involved in prophenoloxidase activation in other insects. Both CLIPs are expressed in mosquito hemocytes and are transcriptionally induced by bacterial and Plasmodium challenges. Functional studies applying RNA interference revealed that both CLIPs are involved in the killing of Plasmodium ookinetes in Anopheles. Studies on parasite melanization demonstrated an additional role for CLIPB14 in the prophenoloxidase cascade. We further report that both CLIPs participate in defense toward Gram-negative bacteria. Our findings strongly suggest that clip domain serine proteases serve multiple functions and play distinctive roles in several immune pathways of A. gambiae.

    Funded by: NIAID NIH HHS: AI044220-07

    The Journal of biological chemistry 2005;280;48;40161-8

  • Fusion tags and chaperone co-expression modulate both the solubility and the inclusion body features of the recombinant CLIPB14 serine protease.

    Schrödel A, Volz J and de Marco A

    EMBL, Meyerhofstr. 1, D-69117 Heidelberg, Germany.

    Chaperone co-expression and the fusion to different tags were used to modify the aggregation pattern of the putative serine protease CLIPB14 precipitated in Escherichia coli inclusion bodies. A set of common tags used in expression vectors has been selected, as well as two bacterial strains over-expressing the chaperones GroELS and ibpA/B, respectively. The presence of the fused tags resulted in an improved solubility of CLIPB14 but also in a higher presence of contaminants in the inclusion bodies, while chaperone co-expression promoted the binding of all the chaperone machinery involved into the disaggregation to the CLIPB14. Furthermore, each tag influenced in a specific manner the re-aggregation of the denatured CLIPB14 constructs during urea dilution and the preliminary trials indicated that the CLIPB14 fusions with higher homogeneity and lower re-aggregation rate were the optimal candidates for refolding assays. In conclusion, it is possible to tune the quality of the inclusion bodies by choosing the suitable combination of tag and chaperone co-expression that minimize the non-productive side reactions during refolding.

    Journal of biotechnology 2005;120;1;2-10

  • Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.

    Niaré O, Markianos K, Volz J, Oduol F, Touré A, Bagayoko M, Sangaré D, Traoré SF, Wang R, Blass C, Dolo G, Bouaré M, Kafatos FC, Kruglyak L, Touré YT and Vernick KD

    Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010, USA.

    Successful propagation of the malaria parasite Plasmodium falciparum within a susceptible mosquito vector is a prerequisite for the transmission of malaria. A field-based genetic analysis of the major human malaria vector, Anopheles gambiae, has revealed natural factors that reduce the transmission of P. falciparum. Differences in P. falciparum oocyst numbers between mosquito isofemale families fed on the same infected blood indicated a large genetic component affecting resistance to the parasite, and genome-wide scanning in pedigrees of wild mosquitoes detected segregating resistance alleles. The apparently high natural frequency of resistance alleles suggests that malaria parasites (or a similar pathogen) exert a significant selective pressure on vector populations.

    Science (New York, N.Y.) 2002;298;5591;213-6

  • Immunity-related genes and gene families in Anopheles gambiae.

    Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Müller HM, Osta MA, Paskewitz SM, Reichhart JM, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P and Kafatos FC

    European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

    We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

    Science (New York, N.Y.) 2002;298;5591;159-65

* quick link - http://q.sanger.ac.uk/mal-ray