Arbeitgruppe: Gilberger group (top left to bottom right): Arne Alder, Dr. Paul-Christian Burda, Louisa Wilcke, Dorothee Heincke, Annika Schmidke, Sarah Scharf, Michael Geiger, Sarah Lemcke, Prof. Dr. Tim-Wolf Gilberger, Dr. Janis Rambow
Gilberger group (top left to bottom right): Arne Alder, Dr. Paul-Christian Burda, Louisa Wilcke, Dorothee Heincke, Annika Schmidke, Sarah Scharf, Michael Geiger, Sarah Lemcke, Prof. Dr. Tim-Wolf Gilberger, Dr. Janis Rambow

Overview

Over a third of the world's population is at risk of malaria, with approximately 300 million people developing clinical disease each year. Transmission of the pathogen, the protozoan parasite Plasmodium spp., occurs during feeding of the Anopheles mosquito when the parasite enters the human circulation and invades liver and red blood cells.

The invasion and subsequent modification of the red blood cells is a basic but crucial step for the survival and multiplication of this deadly parasite. After initial attachment of the parasite to the surface of the target cell, the intruder establishes a tight junction between its apical end and the host cell membrane. This tight junction progressively moves towards the posterior of the invading parasite as it enters the target cell. The process of invasion of red blood cells involves an array of proteins located in specialized exocytic organelles (micronemes, rhoptries and dense granula). They are involved in recognition, adhesion and active invasion of the respective host cell.

After invasion of the red blood cell the malaria parasites (called in this phase ring stages) go through a phase of slow growth. However, these stages do most certainly not lie dormant: the appearance of elaborate parasite-induced modifications in the host cell towards the end of the ring stage, combined with the following onset of rapid growth and multiplication, indicates that this stage serves P. falciparum to prepare the host cell for parasite development. This process of modifying the red blood cell has to occur beyond the parasites' own cellular boundaries and is not only intriguing in cell biological terms, but also likely to have no precedent in mammalian biology.

Figure 1 - Transmission of the parasite ("sporozoites") occurs during feeding of the Anopheles mosquito. The sporozoites migrate through the dermis into the human blood vessels. The first initial multiplication step takes place in hepatocytes of the liver. So called "merozoites" are released from "merosomes" into the bloodstream where they invade erythrocytes and multiply within 48 h. Within the erythrocytes they undergo substantial morphological changes that can be divided into three distinct stages: ring-, trophozoite- and schizont-stages. After schizogony, up to 32 new merozoites are released from destroyed erythrocytes. These merozoites re-invade new erythrocytes and initiate the exponential growth of the parasite. Within weeks after infection some of blood stage parasites differentiates into pre-sexual stages (so called "gametocytes") and are taken up by the mosquito during feeding (schematic by BNITM/ Klaus Jürries)

Our work group focuses on red blood cell invasion of the malaria parasite. Currently, we have four main foci:

  • Regulation of host cell invasion (e.g. phosphorylation and dephosphorylation)
  • Investigations of structural compartments associated with host cell invasion such as the “Inner Membrane Complex” or the secretory organelles
  • Compartment specific protein recruitment
  • Identification and validation of protein networks that power host cell invasion

Research Projects

Red blood cell invasion of the malaria parasite

One of the most significant steps in the complex malaria life cycle is the invasion of human erythrocytes – a step that is crucial and mandatory for its massive multiplication in the human system. All clinical symptoms are connected with the modification and destruction of this host cell. The invasion process is powered by an unknown number of proteins mediating cell adhesion and motility. We study this molecular basis for erythrocyte invasion by combining genetic, cellular, biochemical, structural and systems biology based approaches with the aim to deliver a detailed molecular blueprint that will help to define novel therapeutical targets.

Figure 2 - Approaches to dissect red blood cell invasion of the malaria parasite. In order to understand the molecular machinery the parasite uses to enter erythrocytes we use (A) video and 4 D microscopy , (B) systems biology, (C) molecular biology and (D) structural biology approaches.

Adhesive and regulatory elements

To survive and multiply the parasite invades human red blood cells in less than a minute. It relies on an orchestrated cascade of molecular interactions that is driven by the parasite. The physical link between the parasite and erythrocyte membrane is generated by the interaction of parasite proteins that bind with their adhesive, extracellular domain to specific surface structures of the erythrocyte. This physical bridge between parasite and its host cell is linked to the actin-myosin motor of the parasite powering the invasion. The assembly of these complexes, the engagement of the motor units and the subsequent disassembly of the functional units after successful invasion is tightly regulated. One cellular control mechanism is the post-translational modification of proteins due to phosphorylation.

We are investigating kinase dependent phosphorylation of cytoplasmic domains of type I invasins as a switch mechanism in the molecular cascade triggering and powering the invasion process of human red blood cells. We are interested in the identification of the responsible kinases (and phosphatases) as well as in the dissection of signaling and effector pathways mediated by the cytoplasmic domain of selected invasins.

We are also interested in the function of protein palmitoylation during parasite maturation and invasion. This modification has become increasingly recognized to be of major importance for better understanding of how subcellular localization, complex formation and enzymatic activity of proteins are regulated. By doing so, we can contribute towards a detailed molecular understanding of host cell invasion and deliver mechanistic insights that can be useful for translational approaches.

Figure 2 - Time-lapse microscopy of the invasion of a red blood cell by the malaria parasite
Figure 3 - Time-lapse microscopy of the invasion of a red blood cell by the malaria parasite

Structural elements

Crucially, to transfer myosin's driving force into anterograde movement for host cell invasion of the malaria parasite, the motor complex is anchored in the membranes of the inner membrane complex (IMC) that is located under the parasites plasma membrane in the malaria parasite. The IMC is composed of two closely aligned membranes underlying the parasites plasma membrane. The IMC is one of the traits shared by organisms now recognized as a super group called Alveolata, incorporating the traditional phyla of Ciliates, Apicomplexans and Dinoflagellates.

During evolution the structural role of the IMC was "custom-tailored" for the individual ecological niches of different clades. For apicomplexans, the IMC has three major functions: i) it plays a major role in motility, invasion and egress; ii) it confers stability and shape to the cell and iii) it provides a scaffolding framework during cytokinesis. The obvious fundamental role of the IMC stands in contrast to our rudimentary knowledge of its components, dynamics and biogenesis in the malaria parasite.

We explored a systems biological approach and subsequent phylogenetic profiling to identify novel IMC proteins in the malaria parasite. We revealed high levels of diversity in terms of structural organization and phylogenetic trajectories of Plasmodium IMC proteins, which exemplifies the adaptive molecular composition of this structure. Using high resolution and time-lapse microscopy we investigated i) the dynamic of this structure during parasite maturation, ii) its role in pre-sexual differentiation and iii) its sub-compartimentalization. The latter is exemplified by recent work on a dynamic ring structure, referred to as the basal complex that is part of the IMC and helps divide organelles and abscises the maturing daughter cells.

Beside the IMC we are also interested in the biogenesis and protein composition of the secretory organelles of the parasite given that they are essential cellular structures and responsible for storing and secreting dozens of proteins mediating host cell invasion.

Figure 4 - Biogenesis, composition, dynamic and function of the inner membrane complex. The Golgi dervied IMC is a unique feature of some single cell organisms such as the apicomplexan parasites. It underlies the plasma membrane and is assembled prior to the final nuclear division at the apical pole of nascent daughter cells. It plays an essential role in cytokinesis, cell architecture and motility. (A) The last nuclear (NU) division during the schizogony is accompanied with IMC biogenesis and elongation. The basal rim of the IMC represents the basal complex (BC) in the malaria parasite P. falciparum. (B) Using confocal microscopy the basal complex can be highlighted using the novel marker protein PfBTP1 (green, PfBTP1-GFP). PfBTP1 is closely associated with the plasma membrane (marked with PfSMS1-mCherry), which is wrapped around the daughter cells by plasma membrane invagination. (C) The basal complex moves as a contractile ring from the apical to the basal pole during the last nuclear division. It helps to divide organelles and abscises the maturing daughter cells.

Global networks

Erythrocyte invasion is an active, parasite driven cell intrusion process that cannot be accomplished by single, isolated functional units. It is the result of the interplay of a complex protein network. The Bozdech laboratory (NTU, Singapore) constructed a high confidence gene interactome network. Using the assembled interactome network, we identified a sub-network of proteins that are associated with merozoite invasion by retrieving 418 predicted proteins directly linked to previously established invasion associated proteins


Figure 5 - Blueprint of the protein network implicated in merozoite invasion defining the "invadome". This subnetwork has a total of 2,417 links (purple lines) that are derived from the 90% confidence network and link the 25 reference genes to 25 core apical proteins (marked with red circles) with 418 proteins (Hu and Cabrera et al., 2010).

The functional diversity of the annotated proteins within this invasion subnetwork ("the invadome"), including protein kinases (like CDPK1, PKA), proteases and phosphatases, illustrates the complex machinery that powers erythrocyte invasion. Using a GFP-tagging approach, we selected 70 proteins for experimental analysis of their predicted association with invasion. 42 proteins could be localized in the parasite, of which 31 were targeted either to the apical organelles, the parasite surface or the IMC, compartments directly linked to the invasion process. Some of these proteins revealed distinctive functional domains linking these proteins to proteolysis, protein phosphorylation, adhesion or cytoskeleton interaction, but most of them are hypothetical proteins.

Using reverse genetics, cell biological and biochemical approaches we are now study the function of selected individual proteins in the invadome and expand our localization approach. Likewise, we are working on the re-definition of the invadome by generating and integrating additional global data sets in order to re-calculate the parasite interactome. The experimental validation of the invasion-related subnetwork will increase the resolution of the invadome and will deliver a  blueprint of invasion.

Publications

Abteilung Zelluläre Parasitologie
2017

PfCDPK1 mediated signaling in erythrocytic stages of Plasmodium falciparum
Kumar S, Kumar M, Ekka R, Dvorin JD, Paul AS, Madugundu AK, Gilberger T, Gowda H, Duraisingh MT, Keshava Prasad TS, Sharma P.
Nat Commun. 2017 Jul 5;8(1):63. doi: 10.1038/s41467-017-00053-1.

Arbeitsgruppe Gilberger
2016

A MORN1-associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding
Engelberg K, Ivey FD, Lin A, Kono M, Lorestani A, Faugno-Fusci D, Gilberger TW, White M, Gubbels MJ.
Cell Microbiol. 2016 Feb 3. doi: 10.1111/cmi.12574

Pellicle formation in the malaria parasite
Kono M, Heincke D, Wilcke L, Wong TW, Bruns C, Herrmann S, Spielmann T, Gilberger TW.
J Cell Sci. 2016 Feb 15;129(4):673-80. doi: 10.1242/jcs.181230

Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites
Prinz B, Harvey KL, Wilcke L, Ruch U, Engelberg K, Biller L, Lucet I, ErkelenzS, Heincke D, Spielmann T, Doerig C, Kunick C, Crabb BS, Gilson PR, Gilberger TW.
Sci Rep. 2016 Oct 4;6:34479. doi: 10.1038/srep34479

Arbeitsgruppe Gilberger
2015

Critical Steps in Protein Export of Plasmodium falciparum Blood Stages
Spielmann T, Gilberger TW.
Trends Parasitol. 2015 Oct;31(10):514-25. doi: 10.1016/j.pt.2015.06.010

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans
Paul AS, Saha S, Engelberg K, Jiang RH, Coleman BI, Kosber AL, Chen CT, Ganter M, Espy N, Gilberger TW, Gubbels MJ, Duraisingh MT.
Cell Host Microbe. 2015 Jul 8;18(1):49-60. doi: 10.1016/j.chom.2015.06.003

The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite
Wetzel J, Herrmann S, Swapna LS, Prusty D, John Peter AT, Kono M, Saini S, Nellimarla S, Wong TW, Wilcke L, Ramsay O, Cabrera A, Biller L, Heincke D, Mossman K, Spielmann T, Ungermann C, Parkinson J, Gilberger TW.
J Biol Chem. 2015 Jan 16;290(3):1712-28. doi: 10.1074/jbc.M114.598094

Arbeitsgruppe Gilberger
2014

Regulation of Plasmodium falciparum development by calcium-dependent protein kinase 7 (PfCDPK7)
Kumar P, Tripathi A, Ranjan R, Halbert J, Gilberger T, Doerig C, Sharma P.
J Biol Chem. 2014 Jul 18;289(29):20386-95. doi: 10.1074/jbc.M114.561670

Arbeitsgruppe Gilberger
2013

Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation
Tomlins AM, Ben-Rached F, Williams RA, Proto WR, Coppens I, Ruch U, Gilberger TW, Coombs GH, Mottram JC, Müller S, Langsley G.
Autophagy. 2013 Oct;9(10):1540-52. doi: 10.4161/auto.25832

Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export
Heiber A, Kruse F, Pick C, Grüring C, Flemming S, Oberli A, Schoeler H, Retzlaff S, Mesén-Ramírez P, Hiss JA, Kadekoppala M, Hecht L, Holder AA, Gilberger TW, Spielmann T.
PLoS Pathog. 2013;9(8):e1003546. doi: 10.1371/journal.ppat.1003546

The apicomplexan inner membrane complex
Kono M, Prusty D, Parkinson J, Gilberger TW.
Front Biosci (Landmark Ed). 2013 Jun 1;18:982-92.

PfSec13 is an unusual chromatin-associated nucleoporin of Plasmodium falciparum that is essential for parasite proliferation in human erythrocytes
Dahan-Pasternak N, Nasereddin A, Kolevzon N, Pe'er M, Wong W, Shinder V, Turnbull L, Whitchurch CB, Elbaum M, Gilberger TW, Yavin E, Baum J, Dzikowski R.
J Cell Sci. 2013 Jul 15;126(Pt 14):3055-69. doi: 10.1242/jcs.122119

Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum
Engelberg K, Paul AS, Prinz B, Kono M, Ching W, Heincke D, Dobner T, Spielmann T, Duraisingh MT, Gilberger TW.
Biochem J. 2013 Jun 15;452(3):457-66. doi: 10.1042/BJ20121694

The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion
Mueller C, Klages N, Jacot D, Santos JM, Cabrera A, Gilberger TW, Dubremetz JF, Soldati-Favre D.
Cell Host Microbe. 2013 Mar 13;13(3):289-301. doi: 10.1016/j.chom.2013.02.001

H2A.Z/H2B.Z double-variant nucleosomes inhabit the AT-rich promoter regions of the Plasmodium falciparum genome
Hoeijmakers WA, Salcedo-Amaya AM, Smits AH, Françoijs KJ, Treeck M, Gilberger TW, Stunnenberg HG, Bártfai R.
Mol Microbiol. 2013 Mar;87(5):1061-73. doi: 10.1111/mmi.12151

Arbeitsgruppe Gilberger
2012

Time-lapse imaging of red blood cell invasion by the rodent malaria parasite Plasmodium yoelii
Yahata K, Treeck M, Culleton R, Gilberger TW, Kaneko O.
PLoS One. 2012;7(12):e50780. doi: 10.1371/journal.pone.0050780

Uncovering common principles in protein export of malaria parasites
Grüring C, Heiber A, Kruse F, Flemming S, Franci G, Colombo SF, Fasana E, Schoeler H, Borgese N, Stunnenberg HG, Przyborski JM, Gilberger TW, Spielmann T.
Cell Host Microbe. 2012 Nov 15;12(5):717-29. doi: 10.1016/j.chom.2012.09.010

Shape-shifting gametocytes: how and why does P. falciparum go banana-shaped?
Dixon MW, Dearnley MK, Hanssen E, Gilberger T, Tilley L.
Trends Parasitol. 2012 Nov;28(11):471-8. doi: 10.1016/j.pt.2012.07.007

Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite
Cabrera A, Herrmann S, Warszta D, Santos JM, John Peter AT, Kono M, Debrouver S, Jacobs T, Spielmann T, Ungermann C, Soldati-Favre D, Gilberger TW.
Traffic. 2012 Oct;13(10):1335-50. doi: 10.1111/j.1600-0854.2012.01394.x

Regulation of Plasmodium falciparum glideosome associated protein 45 (PfGAP45) phosphorylation
Thomas DC, Ahmed A, Gilberger TW, Sharma P.
PLoS One. 2012;7(4):e35855. doi: 10.1371/journal.pone.0035855

Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite
Kono M, Herrmann S, Loughran NB, Cabrera A, Engelberg K, Lehmann C, Sinha D, Prinz B, Ruch U, Heussler V, Spielmann T, Parkinson J, Gilberger TW.
Mol Biol Evol. 2012 Sep;29(9):2113-32. doi: 10.1093/molbev/mss081

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing
Deshmukh AS, Srivastava S, Herrmann S, Gupta A, Mitra P, Gilberger TW, Dhar SK.
Nucleic Acids Res. 2012 Jul;40(12):5313-31. doi: 10.1093/nar/gks202

Arbeitsgruppe Gilberger
2011

Highly co-ordinated var gene expression and switching in clinical Plasmodium falciparum isolates from non-immune malaria patients
Bachmann A, Predehl S, May J, Harder S, Burchard GD, Gilberger TW, Tannich E, Bruchhaus I.
Cell Microbiol. 2011 Sep;13(9):1397-409. doi: 10.1111/j.1462-5822.2011.01629.x

A research agenda for malaria eradication: basic science and enabling technologies
Amino R, Bassat Q, Baum J, Billker O, Bogyo M, Bousema T, Christophides G, Deitsch K, Dinglasan R, Djimde A, Duraisingh M, Dzinjalamala F, Happi C, Heussler V, Kramarik J, de Koning-Ward T, Lacerda M, Laufer M, Lim P, Llinas M, McGovern V, Martinez-Barnetche J, Mota MM, Mueller I, Okumu F, Rasgon J, Serazin A, Sharma P, Sinden R, Wirth D, Gilberger T.
PLoS Med. 2011 Jan 25;8(1):e1000399. doi: 10.1371/journal.pmed.1000399

Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions
Grüring C, Heiber A, Kruse F, Ungefehr J, Gilberger TW, Spielmann T.
Nat Commun. 2011 Jan 25;2:165. doi: 10.1038/ncomms1169

Arbeitsgruppe Gilberger
2010

H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3
Bártfai R, Hoeijmakers WA, Salcedo-Amaya AM, Smits AH, Janssen-Megens E, Kaan A, Treeck M, Gilberger TW, Françoijs KJ, Stunnenberg HG.
PLoS Pathog. 2010 Dec 16;6(12):e1001223. doi: 10.1371/journal.ppat.1001223

Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite
Leykauf K, Treeck M, Gilson PR, Nebl T, Braulke T, Cowman AF, Gilberger TW, Crabb BS.
PLoS Pathog. 2010 Jun 3;6(6):e1000941. doi: 10.1371/journal.ppat.1000941

Differential effects of C3d on the immunogenicity of gene gun vaccines encoding Plasmodium falciparum and Plasmodium berghei MSP1(42)
Weiss R, Gabler M, Jacobs T, Gilberger TW, Thalhamer J, Scheiblhofer S.
Vaccine. 2010 Jun 17;28(28):4515-22. doi: 10.1016/j.vaccine.2010.04.054

Plasmodium knowlesi: cause of naturally acquired malaria in humans
Schottelius J, Gilberger T, Ehrhardt S, Burchard G.
Dtsch Med Wochenschr. 2010 Feb;135(7):297-300. doi: 10.1055/s-0029-1244852

Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum
Hu G, Cabrera A, Kono M, Mok S, Chaal BK, Haase S, Engelberg K, Cheemadan S, Spielmann T, Preiser PR, Gilberger TW, Bozdech Z.
Nat Biotechnol. 2010 Jan;28(1):91-8. doi: 10.1038/nbt.1597

Arbeitsgruppe Gilberger
2009

Caught in action: mechanistic insights into antibody-mediated inhibition of Plasmodium merozoite invasion
Treeck M, Tamborrini M, Daubenberger CA, Gilberger TW, Voss TS.
Trends Parasitol. 2009 Nov;25(11):494-7. doi: 10.1016/j.pt.2009.07.008

A novel Plasmodium falciparum erythrocyte binding protein associated with the merozoite surface, PfDBLMSP
Wickramarachchi T, Cabrera AL, Sinha D, Dhawan S, Chandran T, Devi YS, Kono M, Spielmann T, Gilberger TW, Chauhan VS, Mohmmed A.
Int J Parasitol. 2009 Jun;39(7):763-73. doi:10.1016/j.ijpara.2008.12.004

Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process
Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, Spielmann T, Gilberger TW.
PLoS Pathog. 2009 Mar;5(3):e1000322. doi: 10.1371/journal.ppat.1000322

Alumni

Doctoral Thesis

Johanna Wetzel (2012-2014) : Sequence specific recruitment of proteins to the inner membrane complex of the malaria parasite Plasmodium falciparum (Welch, 1897)

Dipto Sinha (2010-2013) : Molecular characterization of membrane associated inner membrane complex proteins in Plasmodium falciparum (Welch, 1897)

Boris Prinz (2010-2013) : Funktionelle Analyse des "Apikalen Membran Antigens 1" (AMA1): Untersuchung zur Rolle der Phosphorylierung des Vakzinkandidaten im Malariaerreger Plasmodium falciparum (Welch, 1897)

Klemens Engelberg (2009-2012) : Characterization of adhesion ligand phosphorylation in the malaria parasite Plasmodium falciparum (Welch, 1897)

Ulrike Ruch (2008-2011) : Charakterisierung des Proteintransports im Malariaerreger P.falciparum : (Welch, 1897)

Maya Kono (2007-2010) : Molekulare Charakterisierung der Invasionsmaschinerie des Malariaerregers Plasmodium falciparum (Welch, 1897)

Ana Cabrera (2007-2010) : Global screening and characterization of Plasmpdium falciparum (Welch, 1897) merozoite putative invasion-related proteins

Susann Herrmann (2006-2009) : Charakterisierung des anterograden Proteintransports im Malariaerreger Plasmodium falciparum (Welch, 1897)

Moritz Treeck (2006-2009) : Transport und Funktion adhäsiver Proteine des Malariaerregers Plasmodium falciparum (Welch, 1897)

Silvia Haase (2004-2008) : Invasion und Modifikation von Erythrozyten durch den Malariaparasiten Plasmodium falciparum (Welch, 1897)

Nicole Struck (2003-2007) : Analyse des zielgerichteten Proteintransports in den Apikoplasten von Plasmodium falciparum (Welch, 1897)

Diploma and Master Thesis

Arne Alder (2016) :

Benthe Winkelmann (2016) :

Viola Ladenburger (2015) :

Tatianna Wong (2015) :

Kathrin Stamms (2012) :

Ann Kathrin Gelhaus (2011) :

Dominik Warszta (2011) :

Dorothee Heincke (2011) :

Sonja Christin Kühn (2011) :

Marcel Waschow (2010) :

Contact

Prof. Dr. rer. nat. Tim-Wolf Gilberger

Phone: +49 40 42818 (-240)
Fax: +49 40 42818 (-400)
E-Mail: gilberger@bnitm.de


Technical Staff

Sarah Lemcke (-635)

PhD Students

Arne Alder (-241)
Dorothee Heincke (-241)
Louisa Wilcke (-241)
Michael Geiger (-635)

Master Students

Sarah Scharf (-241)


Downloads