Damage of eukaryotic cells by the pore-forming toxin sticholysin II: Consequences of the potassium efflux

Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (all), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by Stll, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon all damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury. (C) 2017 Elsevier B.V. All rights reserved.

Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo

Rodrigo Manuel Gonzalez, Françoise Gisou van der Goot Grunberg

Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

2008

Folding and Structure of the Pore Forming Toxin Aerolysin

Mircea Ioan Iacovache

The first obstacle encountered by a bacterial pathogen once inside the host is the plasma membrane surrounding the target cells. Throughout evolution bacteria has acquired and maintained genes that upon stimulation express proteins capable of damaging the membrane of other cells. Among these proteins pore forming toxins (PFTs) are a major class of bacterial effectors that are upregulated and secreted during bacterial infections. As their name suggests, pore forming toxins are proteins capable of inserting transmembrane pores in the membranes of the target cells which in turn leads to the lysis of the cell and release of nutrients. The mechanism by which the PFTs function during a bacterial attack has been the subject of extensive research over the years. In most cases PFTs are produced by the bacteria as soluble proteins that require the help of specialized secretion mechanisms to arrive as functional proteins in the external milieu. Once secreted by the producing bacteria these proteins diffuse towards the target cell and bind to the target membrane. Once bound to the plasma membrane of target cells they are capable of initiating a series of structural changes that will eventually lead to the conversion of the water-soluble PFT to a membrane inserted channel. The series of events and the characterization of the different structural changes required for a PFT to convert from a water-soluble protein to a membrane inserted channel is the subject of this thesis. Aerolysin, a PFT produced by Aeromonas hydrophilla, is one of the best candidates for a research into the details of the mode of action of bacterial PFTs. This particular PFT is produced by the bacterium as a soluble periplasmic protein and then secreted outside of the bacterium as a fully folded protein with the help of a type II secretion system. Binding to the target cell is achieved through two high affinity binding sites that recognize sugar modifications which are absent in A. hydrophila, a mechanism that insures that the producing cell is not damaged by its own PFT. Once bound to the target cell aerolysin requires proteolytic activation, a step which cleaves a C-terminal peptide (CTP). Activation is achieved using proteases present on the target cell and the removal of the CTP is thought to initiate the sequence of events leading to pore formation. Following activation aerolysin is able to oligomerize forming heptameric ring-like structures which spontaneously rearrange forming a transmembrane beta-barrel through the membrane. My thesis project, focused on the structural changes required in the mode of action of aerolysin, set off trying to identify the aminoacid sequence involved in the formation of the transmembrane beta-barrel. It was long thought that aerolysin would cross the membrane in a porin like fashin, forming a beta-barrel through the plasma membrane, primarily due to the lack of a hydrophobic patch of aminoacids in its sequence. An initial model proposed in the early '90s postulated that the only region that could form the transmembrane beta-barrel was the Domain 4 of the protein. In this model the removal of the CTP in the activation process would unravel the hydrophobic residues required for the beta-barrel formation and insertion. We and others were able to show however that the fourth domain of the protein is not involved directly in the formation of the pore and we identified a conserved loop in the third domain of the protein which is responsible for the formation of the beta-barrel. This loop presents an alternating pattern of hydrophobic and hydrophilic residues, a requirement for the formation of a transmembrane pore with a hydrophobic exterior and a hydrophilic cavity. Our research led us to propose a sequence of events upon insertion of the aerolysin pore in which a rearrangement of the DIII-loops of the seven monomers in the oligomer forms the initial beta-barrel and generates a hydrophobic tip which drives insertion of the structure through the membrane. Once the bilayer has been crossed the hydrophobic tips folds back on the membrane in a rivet like fashion, anchoring the pore. Following the identification of the DIII-loop as the region that forms the transmembrane pore my researched focused on the structural changes leading to the conversion of a water-soluble protein to a membrane inserted oligomer. While removal of the CTP is the key requirement for this conversion, the role of the CTP in aerolysin mode of action and the sequence of events triggered by its removal is not fully understood. Using a combination of in vivo, in vitro and in silico approaches we were able to show that the CTP plays a wider role in the aerolysin mode of action than previously thought. Indeed our research shows that the CTP is initially required for the correct folding of the soluble protein inside the bacterium, acting as a intramolecular chaperone during the folding of aerolysin. Following folding the CTP binds tightly to a hydrophobic pocket in the fourth domain of the protein locking the PFT in its soluble conformation, a role resembling C-terminal intramolecular chaperones previously described for tail spikes of bacteriophages or fiber forming collagen. This research will be continued with a study on the structural changes triggered by the removal of the CTP and their role in oligomerization and pore formation. The main focus of my thesis project has been however the determination of the structure of the oligomeric form of aerolysin. This part of the project is still ongoing and will be discussed in the final chapter of my thesis. Using 2D and 3D crystallography, AFM and modeling we hope to be able to improve our current understanding of the aerolysin heptameric form and the structural changes required in its formation.

EPFL2011

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.