Type III secreted effectors that target mitochondria.

A type III secretion system (T3SS) is used by Gram-negative bacterial pathogens to secrete and translocate a battery of proteins, termed effectors, from the bacteria directly into the host cells. These effectors, which are thought to play a key role in bacterial virulence, hijack and modify the activity of diverse host cell organelles, including mitochondria. Mitochondria-the energy powerhouse of the cell-are important cell organelles that play role in numerous critical cellular processes, including the initiation of apoptosis and the induction of innate immunity. Therefore, it is not surprising that pathogenic bacteria use mitochondrially targeted effectors to control host cell death and immunity pathways. Surprisingly, however, we found that despite their importance, only a limited number of type III secreted effectors have been characterised to target host mitochondria, and the mechanisms underlying their mitochondrial activity have not been sufficiently analysed. These include effectors secreted by the enteric attaching and effacing (A/E), Salmonella and Shigella bacterial pathogens. Here we give an overview of key findings, present gaps in knowledge and hypotheses concerning the mode by which these type III secreted effectors control the host and the bacterial cell life (and death) through targeting mitochondria.

EspH Suppresses Erk by Spatial Segregation from CD81 Tetraspanin Microdomains.

Enteropathogenic Escherichia coli (EPEC) belongs to a group of enteric human pathogens known as attaching-and-effacing (A/E) pathogens, which utilize a type III secretion system (T3SS) to translocate a battery of effector proteins from their own cytoplasm into host intestinal epithelial cells. Here we identified EspH to be an effector that prompts the recruitment of the tetraspanin CD81 to infection sites. EspH was also shown to be an effector that suppresses the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) signaling pathway at longer infection times. The inhibitory effect was abrogated upon deletion of the last 38 amino acids located at the C terminus of the protein. The efficacy of EspH-dependent Erk suppression was higher in CD81-deficient cells, suggesting that CD81 may act as a positive regulator of Erk, counteracting Erk suppression by EspH. EspH was found within CD81 microdomains soon after infection but was largely excluded from these domains at a later time. Based on our results, we propose a mechanism whereby CD81 is initially recruited to infection sites in response to EspH translocation. At a later stage, EspH moves out of the CD81 clusters to facilitate effective Erk inhibition. Moreover, EspH selectively inhibits the tumor necrosis factor alpha (TNF-α)-induced Erk signaling pathway. Since Erk and TNF-α have been implicated in innate immunity and cell survival, our studies suggest a novel mechanism by which EPEC suppresses these processes to promote its own colonization and survival in the infected gut.

EspH interacts with the host active Bcr related (ABR) protein to suppress RhoGTPases.

Enteropathogenic Escherichia coli are bacterial pathogens that colonize the gut and cause severe diarrhea in humans. Upon intimate attachment to the intestinal epithelium, these pathogens translocate via a type III secretion system virulent proteins, termed effectors, into the host cells. These effectors manipulate diverse host cell organelles and functions for the pathogen's benefit. However, the precise mechanisms underlying their activities are not fully understood despite intensive research. EspH, a critical effector protein, has been previously reported to disrupt the host cell actin cytoskeleton by suppressing RhoGTPase guanine exchange factors. However, native host proteins targeted by EspH to mediate these activities remained unknown. Here, we identified the active Bcr related (ABR), a protein previously characterized to possess dual Rho guanine nucleotide exchange factor and GTPase activating protein (GAP) domains, as a native EspH interacting partner. These interactions are mediated by the effector protein's C-terminal 38 amino acid segment. The effector primarily targets the GAP domain of ABR to suppress Rac1 and Cdc42, host cell cytotoxicity, bacterial invasion, and filopodium formation at infection sites. Knockdown of ABR expression abolished the ability of EspH to suppress Rac1, Cdc42. Our studies unravel a novel mechanism by which host RhoGTPases are hijacked by bacterial effectors.

Mitochondrial Targeting of the Enteropathogenic Escherichia coli Map Triggers Calcium Mobilization, ADAM10-MAP Kinase Signaling, and Host Cell Apoptosis.

The ability of diarrheagenic bacterial pathogens, such as enteropathogenic Escherichia coli (EPEC), to modulate the activity of mitogen-activated protein kinases (MAPKs) and cell survival has been suggested to benefit bacterial colonization and infection. However, our understanding of the mechanisms by which EPEC modulate these functions is incomplete. In this study, we show that the EPEC type III secreted effector Map stimulates the sheddase activity of the disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and the ERK and p38 MAPK signaling cascades. Remarkably, all these activities were dependent upon the ability of Map to target host mitochondria, mainly via its mitochondrial toxicity region (MTR). Map targeting of mitochondria disrupted the mitochondrial membrane potential, causing extrusion of mitochondrial Ca2+ into the host cell cytoplasm. We also found that Map targeting of mitochondria is essential for triggering host cell apoptosis. Based on these findings, we propose a model whereby Map imported into mitochondria causes mitochondrial dysfunction and Ca2+ efflux into the host cytoplasm. Since Ca2+ has been reported to promote ADAM10 activation, the acute elevation of Ca2+ in the cytoplasm may stimulate the ADAM10 sheddase activity, resulting in the release of epidermal growth factors that stimulate the ERK signaling cascade. As p38 activity is also Ca2+ sensitive, elevation in cytoplasmic Ca2+ may independently also activate p38. We hypothesize that Map-dependent MAPK activation, combined with Map-mediated mitochondrial dysfunction, evokes mitochondrial host cell apoptosis, potentially contributing to EPEC colonization and infection of the gut.IMPORTANCE Enteropathogenic E. coli (EPEC) is an important human diarrhea-causing bacterium. The pathogenic effects of EPEC largely depend upon its ability to inject a series of proteins, termed effectors, into the host cells. One such effector is the mitochondrion-associated protein (Map). Map has been shown to induce actin-rich projections (i.e., filopodia) on the infected cell surface and activate a Rho GTPase enzyme termed Cdc42. Nonetheless, although most injected Map localizes to host mitochondria, its functions in the mitochondria remain unknown. Here, we show that Map targeting of mitochondria stimulates the disruption of mitochondrial membrane potential to induce Ca2+ efflux into the host cytoplasm. The efflux stimulates the activity of a protein termed ADAM10, which induces activation of a mitogen-activated protein kinase cascade leading to host cell apoptosis. As apoptosis plays a central role in host-pathogen interactions, our findings provide novel insights into the functions of mitochondrial Map in promoting the EPEC disease.