Rogerio Amino, Priscille Brodin, Rut Carballido-Lopez, Maria Teresa Catanese, Jost Enninga (organizer), Friedrich Frischknecht (organizer), Kay Grünewald, Volker Heussler, Frank Lafont, Emmanuel Lemichez, Musa Mhlanga, Serge Mostowy, Agneta Richter-Dahlfors, Kristine Schauer, Spencer Shorte, Bettina Stolp, Sven van Teeffelen, Ger van Zandbergen, Michael Way
by Jost Enninga
7 – 12 April 2014
Summary: Infectious disease is a major cause of morbidity and mortality in our increasingly connected world due to widespread antibiotic resistance and the lack of vaccines against a large number of microbes. Understanding the molecular and cellular events of infection and their effects on the whole body are highly relevant for the rational development of novel drugs, vaccines and identification of novel drug targets. Research on this led to a discipline named “cellular microbiology” that combines microbiology, cell biology and immunology. It has become clear that cellular microbiology requires more quantitative, mainly imaging-based approaches including expertise from physicists and computer scientists to meet its goals. With this seminar, leading specialists from the associated research fields have discussed these current challenges to bridge cellular microbiology with cutting-edge microscopy.
Key words: Host-pathogen interactions, Light microscopy, Electron microscopy, Cellular microbiology, Infection models
The conference started discussing novel paradigms how intracellular bacterial pathogens undermine host cell functions, and how this can be analyzed by cutting edge imaging techniques. Jost Enninga (Institut Pasteur, Paris) opened the first session presenting the imaging of host cell invasion by the bacterial pathogen Shigella flexneri. He addressed cell attachment, uptake, vacuolar rupture and inter- as well as intra-cellular spread. Thanks to single-cell fluorescence reporters it is possible to follow quantitatively the temporal order of both the bacterial behavior as well as the cytosolic immune response in a highly heterogeneous setting. Jost presented evidence that the infected host plays an important role in controlling both the maintenance and the rupture of the vacuole involving a network of RabGTPases. The molecular details could be monitored by large volume correlative light electron microscopy. Priscille Brodin (INSERM, Lille) portrayed the power of high-content screening using drug and siRNA libraries during macrophage infection with Mycobacterium tuberculosis. She tracked the growth of this pathogens within the immune cells as readout. Her work identified novel small molecules with an important antimicrobial activity carrying a huge pharmacological potential. During the second half of her talk, she explained how Mycobacterium ulcerans, the causative agent of Buruli ulcer, takes advantage of the secreted polyketide mycolactone, to induce analgesia during the infection. Intracellular pathogens often hijack the host cytoskeleton, and Serge Mostowy (Imperial College, London) outlined the biology of septins, which can be considered as the fourth component of the host cytoskeleton. Using multimodal imaging technologies, he revealed how septins control the entry and intracellular growth of bacterial pathogens, such as Shigella and Listeria. In addition, he established a novel zebrafish infection model that allows imaging from the subcellular to the full body level during bacterial challenge. With this model, he has been capable to study the interaction of intracellular pathogens with different host cell types including stem cells.
The afternoon session of our first day in Les Treilles focused on quantitative imaging approaches to study host responses to parasitic infections. Ger van Zandbergen (Paul-Ehrlich-Institute) discussed Leishmania major (L. major) infection of human immune cells. His research group has been conducting high-content screens in primary human macrophages. To study progression of infection and its modulation by the innate immune system, he used fluorescently labeled parasites that can be tracked by 3D live cell imaging correlated with high-pressure freezing and focused ion beam scanning electron microscopy (SEM). Their imaging data suggests that apoptotic parasites are the first to enter into host cells. The induction of autophagy in cells infected by dead parasites deceives the immune system to turn off inflammatory responses. Rogerio Amino (Institut Pasteur) presented intravital imaging studies of Plasmodium sporozoite infection in rodents. His lab studies the mechanisms and functions of host cell traversal (CT) of the sporozoites leading to invasion of the liver. Using a fading assay that measures the reduction of cytoplasmic GFP signal upon membrane wounding, he observed locomotion of RFP+ parasites through both Kupffer cells and endothelial cells. Freddy Frischknecht (University of Heidelberg) described inter-disciplinary approaches to measure the motility of individual Plasmodium sporozoites. Building upon in vivo observations showing that malaria sporozoites display different motility patterns in different environments, he outlined the power of novel tools and biophysical assays to understand the link between the shape of the parasite, its adhesion properties and its motility. Using micropillars he demonstrated that the environment rather than chemotaxis drives migration patterns. Reflection interference contrast microscopy (RICM), traction force microscopy (TFM) and optical tweezers enabled his laboratory to measure intricate details of the parasite movements. Volker Heussler (University of Bern) discussed Plasmodium berghei interactions with primary hepatocytes. He investigates strategies of cytosolic immune response evasion induced during the proliferation of merozoites. Utilizing transgenic parasites and fluorescent markers of cellular organelles, he showed that the membranes forming the vesicles in which merozoites are packaged prior to secretion into the liver sinusoids are of cellular origin.
The second day started with presentations on novel technologies to investigate viral infections. To better understand the assembly of viral particles Kay Gruenewald (Oxford University) and Maria Teresa Catanese (King’s College, London) portrayed the importance of electron microscopy for viral cell biology. Kay explained cryo- tomography to resolve unprecedented details of the Herpex Simplex virus particle. Follow up investigations using integrated EM approaches revealed that the herpes virion reveals two functional ‘poles’ that seem to be related to cell entry and virus assembly at an intriguing compartment called the nucleoplasmic reticulum. He demonstrated new emerging possibilities to correlate light cell imaging based on fluorescent tags, including super resolution microscopy, with electron and X-ray cryo-microscopy. Maria Teresa outlined progress on our understanding of Hepatitis C virus cell biology using custom-made EM affinity grids. Preparing highly-purified virions for mass spectrometry, Maria Teresa identified novel players of virus assembly. The underlying principles of this process were investigated by cell-based fluorescent reporter systems for sensitive distinction of individual HCV-infected cells. The power of fluorescence microscopy for the study of viral cell biology was demonstrated by Michael Way (Cancer UK, London). In addition to the screening of his ‘best of’ shots of Vaccinia virus surfing on actin waves and microtubules, Michael showed recent work on vaccinia F11 and A36 proteins that interfere with RhoA signaling and N-WASP-Arp2/3-dependent actin polymerization, respectively. Together his work illustrates how studying viral infection can shed light on signaling, transport and cytoskeleton dynamics of the host cell. Bettina Stolp (University of Heidelberg) presented how an integrative imaging approach at different scales revealed an important HIV-1 immune evasion mechanism. She combined in vitro assays, such as migration in flow chambers and collagen gels, with approaches in vivo, including imaging in zebrafish and intravital multiphoton microscopy in mouse. Her elegant studies showed that the HIV protein Nef interferes with actin remodeling and impairs chemotaxis of primary human T lymphocytes, guided motility of zebrafish primordial germ cells and the homing of primary murine lymphocytes to peripheral lymph nodes.
After introducing the different microbes, the conference switched to a broad analysis of emerging quantitative imaging. Spencer Shorte (Imagopole, Institut Pasteur) is dedicated to enable numerous of mainly biology-trained users the access to cutting edge scientific imaging technologies while developing and improving existing imaging systems. One current focus of his research is phototoxicity that can be especially observed during high-speed, multi-dimensional and super-resolution imaging. A second technique, micro-mirror enhanced microscopic imaging (MEMI-OP), allows the bleaching of a defined 3 dimensional area, using ultrafast micro-mirrors to allow high-speed, multi-dimensional, spatial and angular light control for FRAP and optogenetic experiments. Spencer concluded underlining “Get more out of your photons and be kinder to your biology”. Subsequently, Sven van Teffelen introduced Escherichia coli cell growth and the shape-determining peptidoglycan cell wall. With computational simulations Sven modelled the bacterial cell wall as a network of springs under tension that twist upon shrinkage. The mathematical model could show that chiral proteoglycan orientation was dictated by motion of the bacterial protein MreB, leading to the conclusion that the physical coupling between MreB and the cell-wall synthesis results in the uniform bacterial growth.
Switching from pathogens to bacterial toxins, Emmanuel Lemichez (INSERM, Unviersité de Nice) gave an overview how they catalyze post-translational modifications of host cell substrates. His team has established that a group of toxins induces the opening of large transcellular tunnels in endothelial cells (TEMs), through the direct inhibition of RhoA (Staphylococcus aureus EDIN toxin) or by raising the flux of cyclic-AMP in the absence of RhoA inhibition (adenylate cyclases of Bacillus anthracis or B. pertussis). Rut Carballido-Lopez (INRA, Jouy-en-Josas) compared the bacterial counterparts of eukaryotic actin and tubulin, and employed these cytoskeletal elements to perform many functions, including cell morphogenesis. Experiments using total internal reflection fluorescence microscopy (TIRF) have recently shown a dynamic relation between the bacterial protein MreB and cell wall synthesis. Work using highly inclined laminated optical sheet (HILO) microscopy revealed that YkuR, an essential enzyme of the peptidoglycan precursor biosynthetic pathway, forms diffraction-limited cytoplasmic foci that require MreB. Studying nuclear architecture and spatial organization of chromosomes is important for understanding the regulation of gene expression in human health and disease. Musa Mhlanga (CSIR, South Africa) presented new work illustrating a fundamental role for loop-mediated chromatin contact on the transcription of co-regulated genes in a multigene complex. In this case, genome-editing tools were applied to disrupt contacts between gene loops in a well-characterized multigene complex. In addition, the Mhlanga laboratory has recently performed a high content genome wide screen searching for miRNAs involved in the host response to HIV infection discovering a novel viral apoptosis evasion mechanism.
The conference concluded with the presentation of promising imaging approaches that have a high potential to analyze host-pathogen interactions in an integrated way. Frank Lafont (Institut Pasteur, Lille) showed how to combine the use of atomic force microscopy (AFM), fluorescence and transmission electron microscopy to investigate host-pathogen interactions. Using this approach, he showed the recruitment of membrane and cytoplasmic proteins to the site of adhesion, and in the case of actin with nanoscale resolution. Frank developed this further using AFM to probe the interior of the cell by measuring relative stiffness with a deep probe. He was able to image a stiffer Golgi and showed, for the first time, images of mitochondria in live cells using AFM. Kristine Schauer (Institut Curie, Paris) discussed the generation of normalized cells to study trafficking, signaling and host-pathogen interactions through micropatterning. She could not observe a preferential site of focal invasion by Salmonella, however bacteria moved to the bottom of the cell after internalization. She went on outlining how to use micropatterned cells in high-throughput screenings taking advantage of the possibility to apply statistical analysis to compare subtle differences. Agneta Richter-Dahlfors (Karolinska Institutet, Stockholm) addressed combined transcriptomic analysis, nanotechnology and multimodal imaging to investigate the local and the systemic environment during kidney infection by uropathogenic Escherichia coli. She stressed the importance of in vivo systems to study the interplay of cells and tissues, but also the need to improve in vitro systems that closely mimic the complexity of live animals. Agneta is also developing nanoprobes capable of sensing pH, oxygen, C-reactive protein to be used as real time sensors for local imaging at the site of infection and for monitoring inflammatory systemic responses. She finished her presentation bridging science and art and closed an exciting week about cutting edge microscopy for the study of microbial pathogenesis.