Invasion of host cells is a key feature exploited by a large number of microbial pathogens. Our laboratory follows a multidisciplinary approach to unravel the molecular and cellular basis of key invasion steps. During the last years we have established a number of novel fluorescent microscopic approaches to achieve this goal. We have used our assays to correlate the pathogen behaviour and the host responses in single experiments at cellular and subcellular resolution. We typically use Shigella flexneri, the causative of bacterial dysentery, as model pathogen for assay development. Furthermore, we have also turned to other pathogens, such as Salmonella, Mycobacteria, or EPEC. We have also included the study of different host cell types targeted by the microbes. Currently, we are following three major axes of research as outlined below.

1) Bacterial effector translocation into host cells and the induced responses (Cristina Dias Rodrigues, Amalia Stantzos, Laurent Audry and Jost Enninga)

One strategy employed by a number of pathogens is the secretion of bacterial effector proteins into host cells via sophisticated secretion machineries. The type III secretion system is one such machine resembling a molecular syringe. A particularity of the type III secretion needles is their capacity to translocate bacterial effectors in a single step across three membrane layers- the inner and outer bacteria membranes, and the host plasma membrane. We have set up novel fluorescence-based methodologies to track bacterial effector secretion and translocation into single living host cells. Consequently we (i) compare the secretion kinetics between different effectors, (ii) monitor where secretion occurs, (iii) analyze which host cells are targeted, and (iv) investigate the induced host cellular remodelling at the sites of secretion.

2) Localization of invasive bacteria inside host cells and the targeting of host compartments (Alexandre Bobard, Nora Mellouk, José Santos, Marianne Burbage, and Jost Enninga)

Numerous pathogens induce their uptake into host cells, where they remain either in a vacuole bound compartment (e.g. Salmonella, Legionella), or they are capable to rupture the endocytic vacuole to propagate within the host cytoplasm (e.g. Listeria, Shigella). The overall sequence and hierarchy of the involved steps during host cellular uptake are still poorly understood due to the limited number of approaches that allow dynamic and functional investigations at the single cell level. Using novel assays to track the precise intracellular localization of bacterial pathogens, we are able to study how bacterial factors communicate with host factors to shape the intracellular niche of the pathogens. These studies include comparisons of the targeting of different host cell types, particularly of cells that are involved in the formation of an immune response.

3) Host immune responses and how they are undermined (Soudeh Ehsani, Monica Correia, Juliane Lippmann, Jost Enninga)

The contact and pathogen invasion process trigger a specific host immune response highlighted by the induction of a subset of immune genes. Their induction depends on the behaviour of the pathogens and on their intracellular localization. Common reporters for gene expression, like GFP or luciferase, have the disadvantage that they require maturation- a process that takes time which is difficult to predict. Furthermore, other approaches for gene profiling, like gene chips require the disruption of the cells and collection of transcripts from a large number of cells. To overcome these problems we are developing novel reporter systems that allows gene profiling of specific immune genes in single living cells. We can combine these investigations with our work that tracks the localization of invading bacteria and their physiological behaviour. Furthermore, we investigate the effects of the invading pathogens on the architecture of the host cellular genome. The combination of the results from our different research aims yields integrative information on the invasion strategies employed by the pathogens and on the induced host responses.

Keywords: Bacterial invasion, intracellular niche, host immune response, functionalized microscopic assays

Mounier J., Popoff M., Enninga J., Frame M., Sansonetti P., Tran Van Nhieu G. PloS Pathogens 2009 Jan; 5(1)

Gousset K, Schiff E, Langevin C, Marijanovic Z, Caputo A, Browman DT, Chenouard N, de Chaumont F, Martino A, Enninga J, Olivo-Marin JC, Männel D, Zurzolo C. Nat Cell Biol. 2009 Mar;11(3):328-36

Ray K., Bobard A., Danckaert A., Clair C., Ehsani S., Tang C., Sansonetti P., Tran Van Nhieu G., Enninga J. 2010 Cellular Microbiology 2010. Apr 1;12(4):545-56.