SummaryThe fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.

This paper presents a segmentation and tracking method for quantitative analysis of cell dynamics from in vitro videomicroscopy data. The method is based on parametric active contours and includes several adaptations that address important difficulties of cellular imaging, particularly the presence of low-contrast boundary deformations known as pseudopods, and the occurence of multiple contacts between cells. First, we use an edge map based on the average intensity dispersion that takes advantage of relative background homogeneity to facilitate the detection of both pseudopods and interfaces between adjacent cells. Second, we introduce a repulsive interaction between contours that allows correct segmentation of objects in contact and overcomes the shortcomings of previously reported techniques to enforce contour separation. Our tracking technique was validated on a realistic data set by comparison with a manually defined ground-truth and was successfully applied to study the motility of amoebae in a biological research project.

demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of Factin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.