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Visualizing the Early Events of Shigella Invasion at the Molecular and Cellular Level

Final Report Summary - BACTERIAL INVASION (Visualizing the Early Events of Shigella Invasion at the Molecular and Cellular Level)

Shigellosis (or bacillary dysentery) is one of the most devastating diseases in the world being responsible for 150 million annual cases that lead to 1 million mainly infant deaths in developing countries [1]. The disease consists of bloody diarrhea caused by the invasion of the colonic and rectal mucosa by members of the genus Shigella, a Gram-negative bacteria belonging to the family Enterobacteriaceae. From the four species S. boydii, S. dysenteriae, S. flexneri and S. sonnei, the two latter are responsible for the endemic disease. S. dysenteriae 1 accounts for deadly outbreaks in the developing world because it expresses Shiga toxin, a potent cytotoxin. Shigella is highly contagious being transmitted directly from person to person by hand contact, or indirectly by contaminated food or water. The high infectious capacity and rapid occurrence of multiple resistance to antibiotics, as well as the fact that there is no general vaccine available so far, make prevention and treatment of shigellosis a difficult task [2]. The comprehension of the biological events taking place during Shigella infection and invasion of host cells is crucial for the development of an anti-shigellosis vaccine or prophylactic drug.

Experimentally, Shigella is relatively simple to handle, and a huge number of well-established molecular biology and genetic techniques and procedures can be applied to this pathogen due to its close relation to Escherichia coli. Moreover, Shigella is a valuable model system for the study of host-pathogen interactions because of a number of shared features (such as the type III secretion system) with other Gram-negative pathogens, as Salmonella and Yersinia.

The invasive process of Shigella encompasses several steps:

(i) the capacity of bacteria to enter into epithelial cells by macropinocytosis [3];
(ii) their ability to lyse the membrane of the endocytic vacuole upon uptake to escape into the cytoplasm where unrestricted growth occurs [4]; and
(iii) their ability to move intra- and intercellularly spreading from cell to cell by an actin-dependent process [5].

Our objective was to disclose the highly organised events occurring during Shigella invasion of epithelial host cells (by macropinocytosis) at molecular resolution in 3D through the combination of cell biology, microbiology and powerful imaging approaches. For this purpose, we have been employing correlative light electron microscopy, which allows imaging of the same specimen by fluorescence microscopy and by electron microscopy. Through fluorescence microscopy individual biological events can be detected with fluorescent markers, and then this same event can be investigated via electron microscopy yielding the exact ultra-structural cellular and molecular details. To obtain 3D information, we have also been performing electron tomography together with fluorescence microscopy called correlative light electron tomography.

Through the use of specific fluorescent protein markers that are recruited during Shigella invasion into host cells we have been selecting cells in which the bacteria are actively invading into host cells. These cells have been analysed using alternative electron microscopy techniques, such as scanning electron microscopy, transmission electron microscopy and electron tomography. The acquired data has disclosed the ultra-structure within the cell interior in the bacterial surrounding region. Our observations have allowed us to identify the involvement of a novel pathway within Shigella infection into host cells involving the recruitment of membrane vesicles to the bacterial entry site. Altogether, the results from this project have increased our knowledge on the Shigella invasion process to host cells and will hopefully contribute to the ongoing research effort for the development of an anti-shigellosis vaccine or prophylactic drug.

References:

1. Phalipon A., Sansonetti P. J.: Shigella's ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival? Immunol Cell Biol 2007, 85:119 - 129.

2. Sansonetti P. J., Egile C.: Molecular bases of epithelial cell invasion by Shigella flexneri. Antonie Van Leeuwenhoek 1998, 74:191 - 197.

3. Clerc P., Sansonetti P. J.: Entry of Shigella flexneri into HeLa cells: Evidence for directed phagocytosis involving actin polymerisation and myosin accumulation. Infect Immun 1987, 55:2681 - 2688.

4. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J.: Multiplication of Shigella flexneri within HeLa cells: Lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun 1986, 51:461 - 469.

5. Bernardini M. L., Mounier J., d'Hauteville H., Coquis-Rondon M., Sansonetti P. J.: Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A 1989, 86:3867 - 3871.