Subversion of mitochondria by the invasive bacteria Listeria and Shigella – MITOPATHO

Calcium signals will be studied using fluorescent probes using single cell analysis and real time microscopy. These responses will be simultaneously analyzed with the mitochondrial dynamics, the effects on mitchondrial membrane permeabilization and the release of signals associated with autophagy, inflammation and cell death. Experiments with molecular inhibitors will be performed to elucidate underlying mechanisms.

The analysis of calcium signals indicate that the enteroinvasive bacterium Shigella induces atypical calcium responses during cell invasion, of unusually long durations. The remarkably long duration of these responses is permitted by the reorganization of the actin cytoskeleton at the site of bacterial invasion

The chacacterization of confined and sustained calcium signals at Shigella invasion sites suggest that signaling to mitochondria is also restricted at these sites during the early phases of bacterial invasion.

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Shigella, the causative agent of bacillary dysentery, is a leading cause of casualties linked to diarrhoeal diseases in developing countries, with an estimated number of 100 million cases and 1 million deaths / year. Although relatively infrequent, food-borne infections with Listeria monocytogenes, which causes abortion, encephalitis and meningitis, also have a significant public health impact, because of their high hospitalization and mortality rate, with about 30 % of infected people dying. With the emergence of bacterial strains resistant to all known antibiotics, deciphering mechanisms of bacterial virulence is further justified as an indispensible requisite for the development of alternative anti-infectious agents. Listeria and Shigella are enteroinvasive bacterial pathogens that can invade epithelial cells. Following invasion, both Listeria and Shigella lyse the phagocytic vacuole, replicate intracellularly and disseminate from cell to cell, using different molecular strategies to induce actin-based motility. Both bacterial invasion and dissemination properties are critical for virulence. Like many pathogens, these bacteria were shown to induce Ca2+ signaling during invasion. In recent works, proponents from this proposal have shown that Shigella induces local sustained Ca2+ responses at bacterial entry sites leading to mitochondrial activation. These results indicate that local production of mitochondrial ATP is required to drive the Shigella invasion process, but whether mitochondrial dynamics is affected has not been investigated. In contrast, Listeria was shown by other proponents of this proposal to induce transient fragmentation of the mitochondrial network via Ca2+ influx mediated by the listeriolysin O toxin. While inhibiting mitochondrial fission impairs the efficiency of Listeria invasion/replication, the precise role of mitochondrial function is unclear. The goal of the proposal is to identify the role of mitochondria during the early phases of bacterial infection, using the two invasive pathogens Shigella and Listeria as models. The emphasis will be to analyze local (as opposed to global) Ca2+ responses, since they are expected to determine local activation of mitochondria during bacterial challenge.These two models are complementary: Shigella induces local sustained Ca2+ increases leading to local activation of mitochondria, while Listeria induces global Ca2+ increases and affects mitochondrial dynamics and morphology. In both systems, mitochondria are required for bacterial invasion or replication, but their precise role remains undefined. Which step of the bacterial infectious cell cycle (invasion- vacuolar lysis-intracellular replication - cell-to-cell spreading) is affected is not known and will therefore be the focus of our studies. Identifying a role for mitochondria in one of these steps is particularly important since each of these steps is critical for bacterial virulence and could therefore be targeted separately, leading to novel approaches to counter infection by intracellular pathogens. Also, the role of mitochondria in the coordination of early signals controlling autophagy and inflammatory responses will be investigated. This proposal will benefit from the identification of key molecules and important advances in recent years in the mechanisms controlling mitochondria fusion/fission and mitochondrial Ca2+ levels.In addition to its expected impact on our understanding of the molecular processes governing the pathology of these bacterial pathogens, this program has the potential to lead to the development of strategies to combat specific stages of the infectious process. Beyond bacterial pathogenicity, this program may hence also shed new lights on the functioning of mitochondria, an essential organelle whose dysfunction has been implicated in aging as well as in numerous human diseases, affecting in particular the nervous system and skeletal muscle.