The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Enteropathogenic Escherichia coli EspF is targeted to mitochondria and is required to initiate the mitochondrial death pathway

Enteropathogenic Escherichia coli (EPEC) is a causative agent of infant diarrhoea in developing countries. The EspF protein is the product of the espF gene found on the locus of enterocyte effacement, the key pathogenicity island carried by EPEC and enterohemorrhagic E. coli. EspF is injected from adherent EPEC into host cells via a type III secretion system and was previously shown to induce apoptotic cell death and to be required for disruption of host intestinal barrier function. In this work, we show by immunofluorescence and fractionation studies that EspF is targeted to host mitochondria. The N-terminal region of EspF serves as a mitochondrial import signal and, when expressed within cells, can target hybrid green fluorescent protein to mitochondria. Assessment of mitochondrial membrane potential in infected epithelial cells indicated that EspF plays a role in the mitochondrial membrane permeabilization induced by EPEC infection. Furthermore, EspF was associated with the release of cytochrome c from mitochondria into the cytoplasm and with caspase-9 and caspase-3 cleavage. These findings indicate a role for EspF in initiating the mitochondrial death pathway.     

Viral control of mitochondrial apoptosis

Throughout the process of pathogen-host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. Several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the Bcl-2 family. Moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. Here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus.     

Strong myotoxic activity of Trimeresurus malabaricus venom: Role of metalloproteases

Host answers to pathogen attacks define the course of pathogenic events and decide about the fate of the host organism. Infection with coxsackievirus B3 (CVB3) can induce severe myocarditis and pancreatitis. The interplay between host factors and virus components is crucial for the fate of the infected host. As we have shown before, expression of the pro-apoptotic host protein Siva is significantly increased after CVB3 infection, and infected cells are removed by programmed cell death. Analysis of Siva expressed in Escherichia coli revealed that this protein binds three zinc ions, suggesting a rather complex three-dimensional structure. By screening a human heart cDNA library we found a new interaction partner of Siva. The peroxisomal membrane protein PMP22 may be involved in the host response against CVB3. Previous investigations showed that Siva interacts with the cytoplasmic C-terminus of CD27, a member of the tumor necrosis factor receptor group, and transmits an apoptotic signal. With the help of directed two-hybrid assays we determined the N-terminal part of Siva as the binding region for CD27.     

Externalization of host cell protein kinase C during enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children in developing countries. Protein kinase C (PKC), a serine- and threonine-directed protein kinase, is rapidly activated following EPEC infection and this is accompanied by its translocation to a membrane-bound location where it is tightly bound to phosphatidylserine (PS). EPEC infection causes host cell death, one of whose features is externalization of PS. We hypothesized that externalization of PS would be accompanied by externalization of PKC as well. We report that EPEC infection triggers the externalization of PKC to the outer surface of the host cell. Ecto-PKC remains firmly tethered to the cell but can be released by incubation with peptide or protein substrates for the enzyme. Ecto-PKC is intact and biologically active and able to phosphorylate protein substrates on the surface of the host cell. Phosphorylation of whole EPEC bacteria or EPEC-secreted proteins could not be detected. Externalization of PKC could be reproduced by the combination of an apoptotic stimulus (ultraviolet (UV) irradiation) and phorbol myristate acetate (PMA), a procedure which resulted in externalization of >25% of the total cellular content of PKC-alpha. In the presence of ATP, ecto-PKC inhibited UV-induced cell shrinkage, membrane blebbing, and propidium iodide uptake but not the activation of caspases 3 and 7. This is the first report that expression of an ecto-protein kinase is altered by a microbial pathogen and the first to note that externalization of PKC can accompany apoptosis.     

Enteropathogenic Escherichia coli induces modification of the focal adhesions of infected host cells

Enteropathogenic Escherichia coli (EPEC) is a human-specific pathogen that causes severe diarrhoea in young children. The disease involves intimate interaction between the pathogen and the brush border of enterocytes. During infection, EPEC uses a type III secretion system (TTSS) to inject several proteins into the infected cells, and these effector proteins modify specific processes in the host cell. We show that, upon infection, EPEC induces detachment of the infected host cells from the substratum, modification of focal adhesions (FA) in the infected cells and specific dephosphorylation of focal adhesion kinase (FAK). We also show that EPEC-induced cell detachment is dependent on FAK expression by the infected cells. Finally, we demonstrate that cell detachment, FA modification and FAK dephosphorylation are dependent on functional TTSS in the infecting EPEC. These results suggest that EPEC is using its TTSS to inject protein(s) into the infected cells, which can induce FAK dephosphorylation, as well as FAK-dependent FA modification and cell detachment. These processes are specific and probably play an important role in EPEC virulence.     

The proteins (12 and 15 kDa) isolated from heat‐killed Lactobacillus plantarum L67 induces apoptosis in HT‐29 cells

A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl‐2, Bax and the apoptosis‐mediated proteins [caspase‐8, caspase‐3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT‐29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic‐related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t‐Bid) increased with increasing concentrations of the membrane proteins isolated from heat‐killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl‐2, caspase‐8, caspase‐3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat‐killed L. plantarum L67 induce programmed cell death in HT‐29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT‐29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods. Copyright © 2015 John Wiley & Sons, Ltd.     

Influenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process.     

Tunicamycin and brefeldin a induce in plant cells a programmed cell death showing apoptotic features

Summary The recent identification ofDAD (defender against apoptotic death) gene in plants suggests that the N-linked glycosylation of proteins could be an important control point of plant programmed cell death. In this paper we describe the effects of Tunicamycin, an inhibitor of N-linked protein glycosylation, and Brefeldin A, an inhibitor of protein trafficking from the Golgi apparatus, on sycamore (Acer pseudoplatanus L.) cell cultures. These two chemicals proved able to induce a strong acceleration of the cell death; changes in cell and nucleus morphology; an increase in DNA fragmentation, detectable by a specific immunological reaction; and the presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Moreover, Brefeldin A, but not Tunicamycin, strongly stimulated the production of hydrogen peroxide. These results indicate that also in plants chemicals interfering with the activities of endoplasmic reticulum and of Golgi apparatus strongly induce a form of programmed cell death showing apoptotic features.     

The role of programmed cell death in Plasmodium–mosquito interactions

Many host–parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.     

Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.     

Interaction of enteropathogenicEscherichia coli with host epithelial cells

EnteropathogenicEscherichia coli (EPEC) causes severe diarrhea in young children. Upon infection, EPEC induces the assembly of highly organized pedestal-like actin structures in host epithelial cells. All the EPEC genes that are involved in inducing formation of actin pedestals are located in a unique 35 kbp chromosomal pathogenicity island, termed LEE. These genes include thesep genes that encode components of type III protein secretion system, and genes that encode proteins secreted by this system, theesp genes. This protein secretion system is activated upon contact with the host cell, resulting in increased secretion of Esp proteins. Some of these Esp proteins form the translocation apparatus while others are translocated into the cytoplasm of the host cell. Concerted activity of the LEE genes including theeae, esp and thesep genes is needed to trigger signal transduction in the host cell which results in formation of an actin pedestal. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

What role does pyroptosis play in microbial infection?

Pyroptosis, a type of programmed cell death mediated by gasdermin, is characterized by the swelling and rupture of cells, release of cellular contents and a strong inflammatory response, which is critical for controlling microbial infection. Pattern recognition receptors recognize the intracellular and extracellular pathogenic microbial components and stimulate the organism's inflammatory response by activating the pyroptosis signaling pathway and releasing interleukin-1β (IL-1β), IL-18, and other inflammatory factors to promote pathogen clearance and prevent infection. In the process of continuous evolution, pathogens have developed multiple strategies to modulate the occurrence of pyroptosis and thus enhance their ability to induce disease; that is, the competition between host cells and pathogens controls the occurrence of pyroptosis. Competition can directly affect tissue inflammation outbreaks and even alter cell survival. Studies have shown that various bacterial infections, including Shigella flexneri, Salmonella, Listeria monocytogenes, and Legionella pneumophila, can lead to pyroptosis. Pyroptosis is associated with the occurrence and development of various diseases caused by microbial infection, and the identification of molecules related to the pyroptosis signaling pathway may provide new drug targets for the treatment of related diseases. This study reviews the molecular mechanisms of pyroptosis and the role of pyroptosis in microbial infection-related diseases.     

Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children. EPEC induces the formation of actin pedestal in infected epithelial cells. A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals. We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB. Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB–CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle-forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB–CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB–CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.     

Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation

Enteropathogenic Escherichia coli (EPEC) is able to inject its own receptor, a transmembrane protein called translocated intimin receptor, Tir, into the host epithelial cell. The bacterium then uses an outer membrane protein, intimin, to bind to Tir and remains firmly attached to the host cell surface for the duration of the infection. The bacterium is also able to trigger the rearrangement of several host cell proteins, culminating with the formation of an actin-rich, pedestal-like structure beneath the EPEC adherence site. Although several cytoskeletal proteins are rearranged following EPEC infection, the exact role played by these proteins during pedestal formation remains unknown. We report here that talin, an integrin-binding protein, is recruited by EPEC and associates directly with Tir. By surface plasmon resonance (SPR), the predicted value for the dissociation constant ( K _D) for Tir–talin binding was 1.86 × 10⁻⁷ M. We also demonstrate that microinjection of anti-talin antibodies into HeLa cells resulted in the complete inability to focus actin filaments beneath the attached bacterium. These findings demonstrate that talin is essential for EPEC-induced pedestal formation in infected cells.     

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC ΔespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild‐type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two‐hybrid, proteomics, immunofluorescence and co‐immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC‐mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.     

Feedback effects of host-derived adenosine on enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro . Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo , experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.