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Microbial cell 5(12), 525-544 (2018). doi:10.15698/mic2018.12.659, Published by Shared Science Publ., Graz

Apicomplexa are obligate intracellular parasites that cause several human and veterinary diseases worldwide. In contrast to most intracellular pathogens these protozoans are believed to invade a rather passive host cell in a process, that is, tightly linked to the ability of the parasites to move by gliding motility. Indeed specific inhibitors against components of the gliding machinery and the analysis of knockdown mutants demonstrate a linkage of gliding motility and invasion. Intriguingly, new data show that it is possible to block gliding motility, while host cell invasion still occurs. This suggests that either the current models established for host cell invasion need to be critically revised or that alternative, motor independent mechanisms are in place including a more active role of the host cell that can complement a missing actin-myosin-system. Here we discuss some of the discrepancies that need to be addressed for a better understanding of invasion. © 2013.

Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.

The most prominent vector-transmitted diseases in the first and third world are Lyme disease and malaria, respectively. In both cases the transmitted agents are introduced into the skin from where they eventually disseminate into the blood using active motility. We are interested in deciphering the molecular mechanisms underlying the motility of these pathogens and how they relate to the ultrastructure of the pathogens. Here we provide an overview of the microscopy techniques that we use to achieve these goals.

Recent years have seen tremendous progress in our understanding of malaria parasite molecular biology. To a large extent, this progress follows significant developments in genetic, molecular and chemical tools available to study the malaria parasites and related Apicomplexa, in particular Toxoplasma gondii. One area of major advancement has been in understanding parasite host-cell invasion, a process that utilizes several essential molecular mechanisms that are conserved across the different lifecycle stages. Here, we summarize some of the most recent experimental data that shed light on the events underlying preparation and execution of malaria parasite invasion and how these insights might relate to the development of new antimalarial drugs.

Malaria parasites undergo a complex life cycle between their hosts and vectors. During this cycle the parasites invade different types of cells, migrate across barriers, and transfer from one host to another. Recent literature hints at a misunderstanding of the difference between active, parasite-driven migration and passive, circulation-driven movement of the parasite or parasite-infected cells in the various bodily fluids of mosquito and mammalian hosts. Because both active migration and passive transport could be targeted in different ways to interfere with the parasite, a distinction between the two ways the parasite uses to get from one location to another is essential. We discuss the two types of motion needed for parasite dissemination and elaborate on how they could be targeted by future vaccines or drugs.

A new view into the life of malaria parasites is now possible owing to recent advances in imaging techniques and to the generation of tagged parasites. Insights into how parasites interact with their insect vectors and mammalian hosts have been gained by the study of various parasitic forms in their natural environment. Quantitative analysis of Plasmodium ookinete motility has revealed different modes of motility in parasite invasion of the mosquito gut and the extrusion of invaded gut cells from the epithelium. Similar analysis with Plasmodium sporozoites has revealed the importance of parasite motility in transmission from the mosquito vector to the mammalian host.

Summary The fusion of cell biology with microbiology has bred a new discipline, cellular microbiology, in which the primary aim is to understand host‐pathogen interactions at a tissue, cellular and molecular level. In this context, we require techniques allowing us to probe infection in situ and extrapolate quantitative information on its spatiotemporal dynamics. To these ends, fluorescent light-based imaging techniques offer a powerful tool, and the state-of-the-art is defined by paradigms using so-called multidimensional (multi-D) imaging microscopy. Multi-D imaging aims to visualize and quantify biological events through time and space and, more specifically, refers to combinations of: three (3D, volume), four (4D, time) and five (5D, multiwavelength)-dimensional recordings. Successful multi-D imaging depends upon understanding the available technologies and their limitations. This is especially true in the field of microbiology where visualization of infectious/pathogenic activities inside living host systems presents particular technical challenges. Thus, as multi-D imaging rapidly becomes a common bench tool to the cellular microbiologist, this review provides the new user with some of the necessary technical insight required to get the best from these methods.

The Wiskott-Aldrich syndrome protein family member N-WASP is a key integrator of the multiple signalling pathways that regulate actin polymerization via the Arp2/3 complex [1-4]. Our previous studies have shown that N-WASP is required for the actin-based motility of vaccinia virus and is recruited via Nck and WIP [5, 6]. We now show that Grb2 is an additional component of the vaccinia actin tail-forming complex. Recruitment of Nck and Grb2 to viral particles requires phosphorylation of tyrosine residues 112 and 132 of A36R, the vaccinia actin tail nucleator, respectively. The presence of Grb2 on the virus is also dependent on the polyproline-rich region of N-WASP. The Grb2 pathway alone is therefore unable to nucleate actin tails, as its recruitment requires the prior recruitment of N-WASP by Nck. However, Grb2 does play an important role in actin-based motility of vaccinia, as in its absence, the mean number of actin tails per cell is reduced 2.6-fold. Thus, both Nck and Grb2 act in a cooperative manner to stabilize and/or activate the vaccinia actin-nucleating complex. We suggest that such cooperativity between “primary” and “secondary” adaptor proteins is likely to be a general feature of receptor-mediated signalling.

Considering the rapid advancement of the biomedicalsciences, the molecular and cellular biology of mosthost–parasite interactions remains still largely enigmatic.Some of the best understood parasites are those thatcause malaria, one of the leading causes of death frominfectious diseases throughout human history and stilltoday. This special issue on malaria testifies how muchprogress has been made, but also shows how muchmore remains to be discovered. Not only do we lack aproven rationale for a cheap vaccine, but there are stillmany open questions about the most basic processes ofmalaria parasite biology and how the parasite interactswith its hosts. This leaves plenty of scope for new andfundamental discoveries that would gain immensely fromattracting scientists from other fields and funding agen-cies alike.This issue celebrates the international effort that aimsto understand the molecular and cellular biology of themalaria parasite. This effort has benefited tremendouslyfrom two consecutive European Union research net-works, BioMalPar and EVIMalaR that have spanned thelast decade of research (Waters, 2013). Within thesenetworks over 80 laboratories from Europe, Africa, Indiaand Australia joint together to achieve more and fasterprogress than could have been done by individualefforts. Research focused on different areas such asmolecular cell biology, immunology, systems and vectorbiology. The created networks have fostered a spirit ofcollaboration and have led to several dozens of youngresearchers conducting their doctoral and postdoctoralstudies on malaria. Some of the new findings thatemerged from this research are vigorously discussed atconferences such as the BioMalPar conference, which isbeing held for the 10th time this year at the EuropeanMolecular Biology Laboratory in Heidelberg, Germany.We are convinced that both, the international collabora-tive effort and the BioMalPar conference series will con-tinue in the future.This special issue is thus also a tribute to the manyresearchers that have not only worked hard and openlyexchanged their ideas and findings on sometimes verycompetitive issues but also to those that provided for thefuture by inspiring and training the next generation ofresearchers. Within these pages you will find a showcaseof international work with contributions from colleagues inNorthAmerica, Europe,Asia andAustralia but an ominousabsence of contributions from Africa, where malariacauses most deaths. This single fact highlights theongoing and urgent need for stronger research capacitybuilding right where it is most needed.Therearemyriadinteractionsbetweenthemalariapara-site and its different host cells and tissues and no specialissue would be able to even attempt to portrait them all.Nevertheless, the selection of review and primaryresearch presented here give a good overview of thedivergence of research topics in malaria research andhence the scope of the problem.This research has alwaysbenefited from the use of model organisms from the dis-covery of parasite transmission by Ronald Ross usingbirds and the discovery of the liver stages using monkeysas well as the use of mice for early vaccine studies inthe late 1960s. Therefore, a timely review examines thesuccess and promise of humanized rodent models for thestudy of malaria infection (Kaushansky

Unicellular parasites are of high medical relevance as they cause such devastating diseases as malaria or sleeping sickness. Besides the search for improved treatments, research on these parasites is valuable as they constitute interesting model cells to study basic processes of life. They can also serve as valuable reality checks for our presumed understanding of biological processes that emerge from the study of human or yeast cells, as our common ancestor with many parasites is much older than the one with yeast. But working with parasites can be tricky and time-consuming, if not outright impossible. Here, we focus on examples from imaging studies investigating the transmission of the malaria parasite. Achieving an understanding of the processes important for malaria transmission necessitates different imaging approaches and new molecular and material technologies. The discussed techniques will include in vivo imaging of pathogens in living animals, screening methodologies, and new materials as surrogate 3D environments.

Unicellular parasites are of high medical relevance as they cause such devastating diseases as malaria or sleeping sickness. Besides the search for improved treatments, research on these parasites is valuable as they constitute interesting model cells to study basic processes of life. They can also serve as valuable reality checks for our presumed understanding of biological processes that emerge from the study of human or yeast cells, as our common ancestor with many parasites is much older than the one with yeast. But working with parasites can be tricky and time-consuming, if not outright impossible. Here, we focus on examples from imaging studies investigating the transmission of the malaria parasite. Achieving an understanding of the processes important for malaria transmission necessitates different imaging approaches and new molecular and material technologies. The discussed techniques will include in vivo imaging of pathogens in living animals, screening methodologies, and new materials as surrogate 3D environments.

Apicomplexans are curious single-celled organisms. Belonging to the group of chromalveolates, life for an apicomplexan can be parasitic and some species can cause diseases such as malaria or toxoplasmosis. No apicomplexan is alike, although they share some common features such as being highly polar cells with unique apical organelles. They often change the cells of their metazoan hosts. When they move, apicomplexans do not crawl but glide; when they divide, apicomplexans go through mechanisms matched in cell biological bizarreness only by their names. They undergo schizogony or endodyogeny, processes that are usually not part of a regular molecular cell biology textbook; but they should, as their uniqueness might lead to insights into what proteins and processes are truly essential to make progeny. Here we highlight some of our current knowledge of centrosome and microtubule biology of selected apicomplexan parasites for the yeast and metazoan cell biologist to contemplate.

Neutrophils are the most crucial cells for early defence against infections. When appropriately activated, they can kill obligate intracellular pathogens such as Leishmania. However, once the phagocytotic killing has been evaded, neutrophils can serve as host cells for Leishmania. Parasitized neutrophils were suggested to function as a 'Trojan horse', to transfer Leishmania silently to macrophages. In vivo imaging has contributed a second evasion mechanism. We termed it the 'Trojan rabbit' strategy, whereby parasites escape dying neutrophils to infect macrophages. Here, we discuss the different experimental models used to study neutrophil function in leishmaniasis. We suggest that the capacity of neutrophils to function as an immune evasion target depends on the genetic background of the host and the parasite strain used for the experiments.

Summary Animal skin separates the inner world of the body from the largely hostile outside world and is actively involved in the defence against microbes. However, the skin is no perfect defence barrier and many microorganisms have managed to live on or within the skin as harmless passengers or as disease-causing pathogens. Microbes have evolved numerous strategies that allow them to gain access to the layers underneath the epidermis where they either multiply within the dermis or move to distant destinations within the body for replication. A number of viruses, bacteria and parasites use arthropod vectors, like ticks or mosquitoes, to deliver them into the dermis while taking their blood meal. Within the dermis, successful pathogens subvert the function of a variety of skin resident cells or cells of the innate immune system that rush to the site of infection. In this review several interactions with cells of the skin by medically relevant vector-borne pathogens are discussed to highlight the different ways in which these pathogens have come to survive within the skin and to usurp the defence mechanisms of the host for their own ends.

Pathogens interact with their hosts and host cells in many different ways to achieve successful entry, replication and dissemination. Recent progress in the development of molecular tools and imaging techniques have allowed to gain excit- ing new insights into these interactions on the cellular and molecular level. Often these studies have led to the discoveries of basic cellular mechanisms that the pathogen, be it virus, bacterium, fungus or parasite usurps for its own advantage. In this chapter we present and discuss recent in vivo imaging studies of unicell- ular parasites, emphasizing mainly on malaria parasites. The aim of the chapter is to highlight how simple fluorescent and bioluminescent imaging techniques can provide interesting insights in medically important interactions of these pathogens with their hosts.

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.

The study of pathogens and their interactions with host cells has advanced hand-in-hand with developments in optical microscopy. Whereas microbiology benefits enormously from modern imaging technologies, for example, digital imaging and confocal microscopy, it also presents unique challenges. To overcome these, microbiologists are adept at customising imaging methods, and recently there have been studies using state-of-the-art quantitative imaging methods to probe host-pathogen interactions at the single-cell level. Of particular interest are the studies using combined light and electron microscopy methods, bi-arsenical tetra-cysteine tag labelling and automated image-acquisition and analysis for high-throughput/high-content experimentation. These applications demonstrate how imaging methodologies, adapted for microbiology, continue to open avenues for studies that previously have proven inaccessible.

Optical microscopy in 2 or 3 dimensions allows extensive observations of the motility and morphology of living cells, in culture or in tissue. This leads to an exploding accumulation of imaging data and shifts the bottleneck from data acquisition to data analysis. Manual image analysis is often either impossible or exceedingly time‐consuming and subject to uncontrollable user bias and errors. Computerized methods promise to ensure fast, accurate and reproducible processing, but the basic image analysis functions available in standard commercial software are generally not adapted to the complexity of biological images. For this reason, we develop methods based on active contours, a powerful and flexible technique to segment and track objects, that has become very popular in computer vision research. Here, we describe the main benefits and limitations of active contours for our application, and our efforts to adapt and improve these methods for the analysis of cellular dynamics.

The fusion of cell biology with microbiology has bred a new discipline, cellular microbiology, in which the primary aim is to understand host-pathogen interactions at a tissue, cellular and molecular level. In this context, we require techniques allowing us to probe infection in situ and extrapolate quantitative information on its spatiotemporal dynamics. To these ends, fluorescent light-based imaging techniques offer a powerful tool, and the state-of-the-art is defined by paradigms using so-called multidimensional (multi-D) imaging microscopy. Multi-D imaging aims to visualize and quantify biological events through time and space and, more specifically, refers to combinations of: three (3D, volume), four (4D, time) and five (5D, multiwavelength)-dimensional recordings. Successful multi-D imaging depends upon understanding the available technologies and their limitations. This is especially true in the field of microbiology where visualization of infectious/pathogenic activities inside living host systems presents particular technical challenges. Thus, as multi-D imaging rapidly becomes a common bench tool to the cellular microbiologist, this review provides the new user with some of the necessary technical insight required to get the best from these methods.

A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia