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Interaction between Intracellular Bacterial Pathogens and Host Cell Mitochondria

Anna Spier,1,2,3,4 Fabrizia Stavru,1,2,3,5 and Pascale Cossart1,2,3

1Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France; 2Institut National de la Santé et de la Recherche Médicale, U604, Paris, France; 3Institut National de la Recherche Agronomique, USC2020, Paris, France; 4Bio Sorbonne Paris Cité, Université Paris Diderot, Paris, France; 5Centre National de la Recherche Scientifique, SNC 5101, France.

INTRODUCTION

Mitochondria are dynamic organelles, which are fundamental to eukaryotic cell function. They originated from an endosymbiotic alphaproteobacterium of the genus Rickettsia, which was internalized by the ancestor of all eukaryotes (1). Consistent with this endosymbiotic event, mitochondria are surrounded by a double membrane and still share molecular and morphological features with prokaryotic cells, such as the ability to create energy in the form of ATP through aerobic respiration. To do so, mitochondria oxidize nutrients in a process termed oxidative phosphorylation, which involves the creation and harnessing of a membrane potential across the inner mitochondrial membrane, resulting in ATP synthesis.

Apart from energy production, mitochondria carry out essential steps of heme, iron-sulfur cluster, and ami-no acid biosynthesis as well as fatty acid oxidation and play an important role in calcium homeostasis and cell-autonomous innate immunity (2). In this context, mitochondria display antimicrobial activity through reactive oxygen species (ROS) production and through signaling. Mitochondrial innate immune signaling is mediated by the mitochondrial antiviral signaling protein (MAVS) and results in an interferon response (2, 3). Importantly, mitochondria also play a key role in apoptosis, as the intrinsic apoptosis pathway converges on mitochondrial outer membrane permeabilization (MOMP), which represents a point of no return. Mito-chondrion-mediated apoptosis is highly regulated by members of the B cell lymphoma 2 (Bcl-2) protein family; proapoptotic BH3-only proteins are activated by intracellular stress signals, overcome the inhibitory effect of antiapoptotic Bcl-2 proteins, and enhance recruitment of Bcl2-associated X protein (Bax) and Bcl-2 antagonist or killer (Bak) to the mitochondrial outer membrane. There, Bax and Bak oligomerization results in MOMP and allows the release of cytochrome c, second mitochondrion-derived activator of caspases (SMAC), and Omi, promoting caspase activation and apoptosis (4).

Along with innate immune signaling and apoptosis, the highly dynamic morphology of mitochondria is one of the characteristics of the organelle that clearly differentiate it from most bacteria. Mitochondrial morphology is determined by a steady-state balance between the opposing events of fusion and fission, which are mediated by a set of dynamin-related GTPases. Mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) coordinate outer membrane fusion by homo- and heterotypic interactions, while optic atrophy 1 (Opa1) mediates fusion of the inner membrane. The current model for mitochondrial fission involves initial mitochondrial constriction through the endoplas-mic reticulum (ER) and actin, followed by recruitment of dynamin-related protein 1 (Drp1) to its receptors on the mitochondrial outer membrane. There, Drp1 oligomerizes to form ring-like structures and mediates GTP-dependent mitochondrial fission in conjunction with dynamin 2 (5). Interestingly, Drp1-dependent mitochondrial fission is observed during apoptosis but is not strictly required for its progression (4). Depending on the cell type and functional status of mitochondria, they can adapt their morphology according to cellular energy demands (6) and move along cytoskeletal tracks with the help of molecular motors (7).

Owing to their central role in multiple cellular processes, mitochondria are an attractive target for pathogens. Modulation of mitochondrial functions can be advantageous for bacteria in terms of access to nutrients and/or evasion of the humoral immune system. Here, we explore the relationship between intracellular bacteria and host cell mitochondria, primarily focusing on the effect of the bacteria on mitochondrial morphology and manipulation of host cell death. Manipulation of cell death allows the bacteria to either preserve their intracellular niche by enhancing survival of the host cell or favor dissemination by inducing host cell death. We present examples of both cytosolic and intravacuolar pathogenic bacteria, including Listeria monocytogenes, Shigella flexneri, Rickettsia spp., Legionella pneumophila, Mycobacterium tuberculosis, Salmonella enterica, Chlamydia spp., and Ehrlichia chaffeensis. While cytosolic bacteria are able to directly interact with mitochondria and other organelles, intravacuolar pathogens are confined within a mem-brane-enclosed vacuole and employ specialized secretion systems to introduce effector proteins into the host cell cytoplasm that target mitochondria.

CYTOSOLIC BACTERIA

Listeria monocytogenes

The Gram-positive bacterium L. monocytogenes is a facultative intracellular pathogen causing the foodborne disease listeriosis, which mainly and most severely affects immunocompromised individuals. L. monocytogenes is capable of invading both phagocytic and nonphagocytic cells and employs the phospholipases PlcA and PlcB and the pore-forming toxin listeriolysin O (LLO) to escape the phagosome (8). Inside the cytosol, L. monocytogenes replicates and hijacks the host actin polymerization machinery in order to spread nonlytically to neighboring cells (9). Infection of epithelial cells with L. monocytogenes interferes with mitochondrial dynamics and induces a strong and rapid but transient fragmentation of the mitochondrial network at early time points of infection. The fragmentation is specific to virulent L. monocytogenes, and the secreted toxin LLO has been identified as the causative factor, but the exact mechanism remains to be elucidated. LLO appears not to localize to mitochondria, but rather oligomerizes and forms pores in the plasma membrane, causing a calcium influx, which is crucial for the induction of mitochondrial fission (10) (Fig. 1). Moreover, the L. monocytogenes-induced mitochondrial fragmentation is atypical, as it is independent of Opa1 and Drp1. Indeed, Drp1 dissociates from mitochondria upon infection. On the other hand, the ER and actin, which both have been suggested as regulators of canonical mitochondrial fragmentation, play a role in this type of mitochondrial fission (11).

L. monocytogenes-induced mitochondrial fragmentation is not associated with apoptosis, as classical apoptotic markers such as cytochrome c release and Bax recruitment to mitochondria are absent. Nevertheless, LLO impacts mitochondrial function, since it causes a dissipation of the mitochondrial membrane potential as well as a drop in respiration activity and cellular ATP levels (10). Whether mitochondrial fragmentation directly impacts the host cell metabolic switch to glycolysis (12) remains speculative. As both the mitochondrial network morphology and ATP level recover within a few hours, mitochondria seem not to be terminally damaged upon infection. Interestingly, mitochondrial dynamics plays an important role in L. monocytogenes infection. It was shown that treatment with small interfering RNA favoring mitochondrial fusion augments the infection efficiency, whereas cells with fragmented mitochondria are less susceptible to L. monocytogenes infection. Based on these observations, it was proposed that L. monocytogenes targets mitochondria to temporarily impair mitochondrial functions in order to establish its replication niche (10). Subsequent studies showed that one of the mitochondrial functions, i.e., cell-autonomous innate immune signaling through MAVS, is not active during L. monocytogenes infection, and innate immune signaling is rather mediated by peroxisome-localized MAVS (13).

Shigella flexneri

S. flexneri is a Gram-negative bacterium which causes shigellosis, an inflammatory disease of the colon leading to tissue destruction, and a leading cause of diarrhea in the developing world. After crossing the colonic epithelium, the facultative intracellular pathogen infects both myeloid immune cells and intestinal epithelial cells. S. flexneri injects secreted effectors into the host cell by its type III secretion system (T3SS) to induce membrane ruffling, resulting in enterocyte invasion. The bacterium then rapidly escapes from the phagosome and proliferates in the cytosol, where it employs the host cell actin machinery for intracellular motility as well as for cell-to-cell spread (14). Interestingly, mitochondria were observed at bacterial invasion sites and appear to be entrapped in an actin meshwork induced by the bacterium (15). The authors of the study proposed a model in which an increase in mitochondrial calcium concentration would activate mitochondrial ATP production to locally provide ATP for further actin polymerization (15).

Figure 1 Strategies of intracellular bacteria to interfere with mitochondrial morphology. In epithelial cells, the secreted L. monocytogenes toxin LLO induces rapid mitochondrial fragmentation by pore formation in the plasma membrane, enabling calcium influx. Independent of Drp1 and...