Our research projects at a glance

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.

 

Graphical Abstract
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.

 

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

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.

Blueprint of the protein network
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).

Abteilung Zelluläre Parasitologie

Direktes Portrait von Tim-Wolf Gilberger
Abteilung Zelluläre Parasitologie

Prof. Dr. Tim-Wolf Gilberger

Telefon: +49 40 285380 87-600

E-Mail: gilberger@bnitm.de

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