Sylvia Münter

Rapid control of protein level in the apicomplexan Toxoplasma gondii

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites.

Sporozoites are the highly motile stages of the malaria parasite injected into the hosts skin during a mosquito bite. In order to navigate inside of the host, sporozoites rely on actin-dependent gliding motility. Although the major components of the gliding machinery are known, the spatiotemporal dynamics of the proteins and the underlying mechanism powering forward locomotion remain unclear. Here, we show that sporozoite motility is characterized by a continuous sequence of stick-and-slip phases. Reflection interference contrast and traction force microscopy identified the repeated turnover of discrete adhesion sites as the underlying mechanism of this substrate-dependent type of motility. Transient forces correlated with the formation and rupture of distinct substrate contact sites and were dependent on actin dynamics. Further, we show that the essential sporozoite surface protein TRAP is critical for the regulated formation and rupture of adhesion sites but is dispensable for retrograde capping.

Signaling During Pathogen Infection

Over the millennia, pathogens have coevolved with their hosts and acquired the ability to intercept, disrupt, mimic, and usurp numerous signaling pathways of those hosts. The study of host/pathogen interactions thus not only teaches us about the intricate biology of these parasitic invaders but also provides interesting insights into basic cellular processes both at the level of the individual cell and more globally throughout the organism. Host/pathogen relationships also provide insights into the evolutionary forces that shape biological diversity. Here we review a few recent examples of how viruses, bacteria, and parasites manipulate tyrosine kinase–mediated and Rho guanosine triphosphatase–mediated signaling pathways of their hosts to achieve efficient entry, replication, and exit during their infectious cycles. Pathogens infect almost every living organism. In animals, including humans, the diversity of pathogens ranges from viruses, bacteria, and unicellular parasites to complex fungi, worms, and arthropods. Because pathogens have coevolved with their hosts and have sometimes been coopted as symbionts or commensals, each pathogen/host pair represents a striking success story of survival that reflects the biological complexity of both parties. All invading microorganisms face similar problems, such as gaining access to their host, achieving successful replication, and spreading to a similar or different host. It is therefore not surprising that many different pathogens target similar organs, cell types, and even molecules to achieve their goals. However, no two microbial parasites appear to be completely alike. Although they often target similar signaling networks, they do so in subtly different ways to achieve the desired outcome. This review has eight figures, three movies, and 139 citations and emphasizes two well-established signaling pathways that are often activated during the interaction of different pathogens with their host cells. It illustrates a small part of how the dissection of host/pathogen interactions can reveal, on a molecular scale, a nature shaped by evolutionary forces that can rival the great descriptions of our macroscopic world.

Multistep adhesion of Plasmodium sporozoites

Adhesion of eukaryotic cells is a complex process during which interactions between extracellular ligands and cellular receptors on the plasma membrane modulate the organization of the cytoskeleton. Pathogens particularly rely often on adhesion to tissues or host cells in order to establish an infection. Here, we examined the adhesion of Plasmodium sporozoites, the motile form of the malaria parasite transmitted by the mosquito, to flat surfaces. Experiments using total internal reflection fluorescence microscopy and analysis of sporozoites under flow revealed a stepwise and developmentally regulated adhesion process. The sporozoite‐specific transmembrane proteins TRAP and S6 were found to be important for initial adhesion. The structurally related protein TLP appears to play a specific role in adhesion under static conditions, as tlp(−) sporozoites move 4 times less efficiently than wild‐type sporozoites. This likely reflects the decreased intradermal sporozoite movement of sporozoites lacking TLP. Further, these three sporozoite surface proteins also act in concert with actin filaments to organize efficient adhesion of the sporozoite prior to initiating motility and host cell invasion.−Hegge, S., Munter, S., Steinbüchel, M., Heiss, K., Engel, U., Matuschewski, K., Frischknecht, F. Multistep adhesion of Plasmodium sporozoites. FASEB J. 24, 2222–2234 (2010). www.fasebj.org

Critical Role for Heat Shock Protein 20 (HSP20) in Migration of Malarial Sporozoites

Background: Small heat shock proteins have been associated with microfilament regulation. Results: Ablation of HSP20 impairs the speed, directionality, and adhesion of Plasmodium sporozoites. Conclusion: HSP20 is a key factor for locomotion and infection of the malaria parasite. Significance: This study is the first genetic evidence for a role of a small heat shock protein in cellular motility. Plasmodium sporozoites, single cell eukaryotic pathogens, use their own actin/myosin-based motor machinery for life cycle progression, which includes forward locomotion, penetration of cellular barriers, and invasion of target cells. To display fast gliding motility, the parasite uses a high turnover of actin polymerization and adhesion sites. Paradoxically, only a few classic actin regulatory proteins appear to be encoded in the Plasmodium genome. Small heat shock proteins have been associated with cytoskeleton modulation in various biological processes. In this study, we identify HSP20 as a novel player in Plasmodium motility and provide molecular genetics evidence for a critical role of a small heat shock protein in cell traction and motility. We demonstrate that HSP20 ablation profoundly affects sporozoite-substrate adhesion, which translates into aberrant speed and directionality in vitro. Loss of HSP20 function impairs migration in the host, an important sporozoite trait required to find a blood vessel and reach the liver after being deposited in the skin by the mosquito. Our study also shows that fast locomotion of sporozoites is crucial during natural malaria transmission.

Environmental constraints guide migration of malaria parasites during transmission.

Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission.

Synergistic and additive effects of epigallocatechin gallate and digitonin on Plasmodium sporozoite survival and motility.

Background Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to facilitate absorption of polar agents across cellular membranes. However, little is known about synergistic effects between such potential drug candidates and adjuvants. To probe for such effects, we tested the green tea compound epigallocatechin gallate (EGCG) and the membrane permeabilising digitonin on Plasmodium sporozoite motility and viability. Methodology/Principal Findings Green fluorescent P. berghei sporozoites were imaged using a recently developed visual screening methodology. Motility and viability parameters were automatically analyzed and IC50 values were calculated, and the synergism of drug and adjuvant was assessed by the fractional inhibitory concentration index. Validating our visual screening procedure, we showed that sporozoite motility and liver cell infection is inhibited by EGCG at nontoxic concentrations. Digitonin synergistically increases the cytotoxicity of EGCG on sporozoite survival, but shows an additive effect on sporozoite motility. Conclusions/Significance We proved the feasibility of performing highly reliable visual screens for compounds against Plasmodium sporozoites. We thereby could show an advantage of administering mixtures of plant metabolites on inhibition of cell motility and survival. Although the effective concentration of both drugs is too high for use in malaria prophylaxis, the demonstration of a synergistic effect between two plant compounds could lead to new avenues in drug discovery.

Structural basis for chirality and directional motility of Plasmodium sporozoites

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent‐shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non‐motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo‐electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As aconsequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.

Spotlight on pathogens: 'Imaging Host-pathogen Interactions'.

The self-evidence of pictures has nowadays taken a back seat, as pictures are abundantly present in our world. Only proverbs like ‘seeing is believing’ still acknowledge the predominance of the picture over the written word. While the social sciences are still struggling who should deal with the analysis of the influence of all the pictures on society (Boehm, 1994), the natural sciences already accepted the high impact of pictures on their hypotheses and are waiting for the impact films will have on science. Despite this, we will give a written overview of a symposium held in Heidelberg, which tried to connect the rather new field of cellular microbiology (Cossart et al., 1996) with the latest advances in imaging techniques. Several meetings were already dedicated to this subject over the last years (Lehmann and Frischknecht, 2006; Celli and Knodler, 2008). These clearly showed that pathogens represent not only interesting microscopic objects themselves, but can also be used as probes to decipher complex cell biological processes as they turn host cell functions for their own benefit (Lehmann and Frischknecht, 2006). To successfully image the interactions between pathogens and their host cells an interdisciplinary approach between imaging scientists and microbiologists is required. The symposium in Heidelberg put a strong emphasis on different infection paradigms and on dynamic imaging of infections, as real-time imaging in multiple dimensions is getting widely accessible. Lectures on in silico modelling, image rendering and object tracking completed the programme. Freddy Frischknecht and Maik Lehmann (both from the University of Heidelberg, Germany) welcomed the congress in a charming villa in the old city centre of Heidelberg. The probes – viruses, fungi, parasites, bacteria and prions – seemed almost like a diverse collection out of Pandora’s box. But unlike Pandora, the masters of these beasts found the matching imaging techniques to observe the probes’ fate in detail. Like in a vitreous Pandora’s box, the audience could watch the destructive pathogens unfold their virulence. In a short retrospective on the history of microscopy, Freddy Frischknecht pointed out that the foundation of modern physical optics was actually laid in a book written by the Arab Ibn al-Haytham. Being under house arrest in Cairo from 1011 until 1021, he wrote ‘The Book of Optics’, which influenced the development of that field and especially the emerging understanding of light and vision. About 650 years later the Dutch tradesman Antoni van Leeuwenhoek built many single-lens microscopes and noticed, for example, bacteria on rotten teeth and red blood cells in the vasculature of living fish. Observing for the first time single cells and pathogens with a magnification of more than 200 times, he thus can be considered as the founder of cell and pathogen imaging despite the fact that a formal acceptance of microorganisms as causative agents of disease had to wait another 200 years.