Bacterial toxins are potent virulence factors that have evolved exquisite specificity and efficiency in modulating the host at the cellular level. This Research Topic will highlight recent advances in our understanding of the molecular interactions of toxins with the host, the mechanisms by which toxins promote virulence, and the consequences of these actions on the outcome of infection.

University of Oklahoma Health Sciences Center

University of California, Los Angeles

Views

Article Views

Article Downloads

Frontiers in Cellular and Infection Microbiology

Bacteria and Host

Molecular Bacterial Pathogenesis

Cellular and Infection Microbiology Archive

Critical advances in the study of bacterial toxins

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori). Chronic infection with this Gram-negative pathogen is associated with the development of peptic ulcers and is linked to an increased risk of gastric cancer. H. pylori secretes many proteinaceous factors that are important for initial colonization and subsequent persistence in the host stomach. One of the major protein toxins secreted by H. pylori is the Vacuolating cytotoxin A (VacA). After secretion from the bacteria via a type V autotransport secretion system, the 88 kDa VacA toxin (comprised of the p33 and p55 subunits) binds to host cells and is internalized, causing severe “vacuolation” characterized by the accumulation of large vesicles that possess hallmarks of both late endosomes and early lysosomes. The development of “vacuoles” has been attributed to the formation of VacA anion-selective channels in membranes. Apart from its vacuolating effects, it has recently become clear that VacA also directly affects mitochondrial function. Earlier studies suggested that the p33 subunit, but not the p55 subunit of VacA, could enter mitochondria to modulate organelle function. This raised the possibility that a mechanism separate from pore formation may be responsible for the effects of VacA on mitochondria, as crystallography studies and structural modeling predict that both subunits are required for a physiologically stable pore. It has also been suggested that the mitochondrial effects observed are due to indirect effects on pro-apoptotic proteins and direct effects on mitochondrial morphology-related processes. Other studies have shown that both the p55 and p33 subunits can indeed be efficiently imported into mammalian-derived mitochondria raising the possibility that they could re-assemble to form a pore. Our review summarizes and consolidates the recent advances in VacA toxin research, with focus on the outstanding controversies in the field and the key remaining questions that need to be addressed.

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori). Chronic infection with this Gram-negative pathogen is associated with the development of peptic ulcers and is linked to an increased risk of gastric cancer. H. pylori secretes many proteinaceous factors that are important for initial colonization and subsequent persistence in the host stomach. One of the major protein toxins secreted by H. pylori is the Vacuolating cytotoxin A (VacA). After secretion from the bacteria via a type V autotransport secretion system, the 88 kDa VacA toxin (comprised of the p33 and p55 subunits) binds to host cells and is internalized, causing severe “vacuolation” characterized by the accumulation of large vesicles that possess hallmarks of both late endosomes and early lysosomes. The development of “vacuoles” has been attributed to the formation of VacA anion-selective channels in membranes. Apart from its vacuolating effects, it has recently become clear that VacA also directly affects mitochondrial function. Earlier studies suggested that the p33 subunit, but not the p55 subunit of VacA, could enter mitochondria to modulate organelle function. This raised the possibility that a mechanism separate from pore formation may be responsible for the effects of VacA on mitochondria, as crystallography studies and structural modeling predict that both subunits are required for a physiologically stable pore. It has also been suggested that the mitochondrial effects observed are due to indirect effects on pro-apoptotic proteins and direct effects on mitochondrial morphology-related processes. Other studies have shown that both the p55 and p33 subunits can indeed be efficiently imported into mammalian-derived mitochondria raising the possibility that they could re-assemble to form a pore. Our review summarizes and consolidates the recent advances in VacA toxin research, with focus on the outstanding controversies in the field and the key remaining questions that need to be addressed.

Vacuolating cytotoxin A (VacA), a key toxin for Helicobacter pylori pathogenesis

Enterohemorrhagic Escherichia coli (EHEC) are responsible for major outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) throughout the world. The mortality associated with EHEC infections stems from the production and release of a potent Shiga toxin (Stx) by these bacteria. Stx induces cell death in endothelial cells, primarily in the urinary tract, causing HUS. Stx was first described in Shigella dysenteriae serotype I by Kiyoshi Shiga and was discovered later in EHEC. Multiple environmental cues regulate the expression of Stx, including temperature, growth phase, antibiotics, reactive oxygen species (ROS), and quorum sensing. Currently, there is no effective treatment or prophylaxis for HUS. Because antibiotics trigger Stx production and their use to treat EHEC infections is controversial, alternative therapeutic strategies have become the focus of intense research. One such strategy explores quorum sensing inhibitors as therapeutics. These inhibitors target quorum sensing regulation of Stx expression without interfering with bacterial growth, leading to the hypothesis that these inhibitors impose less selective pressure for bacteria to develop drug resistance. In this review, we discuss factors that regulate Stx production in EHEC, as well as novel strategies to prevent and/or minimize the development of HUS in infected subjects.

Enterohemorrhagic Escherichia coli (EHEC) are responsible for major outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) throughout the world. The mortality associated with EHEC infections stems from the production and release of a potent Shiga toxin (Stx) by these bacteria. Stx induces cell death in endothelial cells, primarily in the urinary tract, causing HUS. Stx was first described in Shigella dysenteriae serotype I by Kiyoshi Shiga and was discovered later in EHEC. Multiple environmental cues regulate the expression of Stx, including temperature, growth phase, antibiotics, reactive oxygen species (ROS), and quorum sensing. Currently, there is no effective treatment or prophylaxis for HUS. Because antibiotics trigger Stx production and their use to treat EHEC infections is controversial, alternative therapeutic strategies have become the focus of intense research. One such strategy explores quorum sensing inhibitors as therapeutics. These inhibitors target quorum sensing regulation of Stx expression without interfering with bacterial growth, leading to the hypothesis that these inhibitors impose less selective pressure for bacteria to develop drug resistance. In this review, we discuss factors that regulate Stx production in EHEC, as well as novel strategies to prevent and/or minimize the development of HUS in infected subjects.

Shiga toxin in enterohemorrhagic E.coli: regulation and novel anti-virulence strategies

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling.

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling.

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

Bacillus anthracis, the causative agent of anthrax, secretes a tri-partite exotoxin that exerts pleiotropic effects on the host. The purification of the exotoxin components, protective antigen, lethal factor, and edema factor allowed the rapid characterization of their physiologic effects on the host. As molecular biology matured, interest focused on the molecular mechanisms and cellular alterations induced by intoxication. Only recently have researchers begun to connect molecular and cellular knowledge back to the broader physiological effects of the exotoxin. This review focuses on the progress that has been made bridging molecular knowledge back to the exotoxin’s physiological effects on the host.

Bacillus anthracis, the causative agent of anthrax, secretes a tri-partite exotoxin that exerts pleiotropic effects on the host. The purification of the exotoxin components, protective antigen, lethal factor, and edema factor allowed the rapid characterization of their physiologic effects on the host. As molecular biology matured, interest focused on the molecular mechanisms and cellular alterations induced by intoxication. Only recently have researchers begun to connect molecular and cellular knowledge back to the broader physiological effects of the exotoxin. This review focuses on the progress that has been made bridging molecular knowledge back to the exotoxin’s physiological effects on the host.

Cellular and Physiological Effects of Anthrax Exotoxin and Its Relevance to Disease

Pathogenic bacteria utilize multiple approaches to establish infection and mediate their toxicity to eukaryotic cells. Dedicated protein machines deposit toxic effectors directly inside the host, whereas secreted toxins must enter cells independently of other bacterial components. Regardless of how they reach the cytosol, these bacterial proteins must accurately identify their intracellular target before they can manipulate the host cell to benefit their associated bacteria. Within eukaryotic cells, post-translational modifications and individual targeting motifs spatially regulate endogenous host proteins. This review focuses on the strategies employed by bacterial effectors to associate with a frequently targeted location within eukaryotic cells, the plasma membrane.

Pathogenic bacteria utilize multiple approaches to establish infection and mediate their toxicity to eukaryotic cells. Dedicated protein machines deposit toxic effectors directly inside the host, whereas secreted toxins must enter cells independently of other bacterial components. Regardless of how they reach the cytosol, these bacterial proteins must accurately identify their intracellular target before they can manipulate the host cell to benefit their associated bacteria. Within eukaryotic cells, post-translational modifications and individual targeting motifs spatially regulate endogenous host proteins. This review focuses on the strategies employed by bacterial effectors to associate with a frequently targeted location within eukaryotic cells, the plasma membrane.

Bacterial Toxin Effector-Membrane Targeting: Outside in, then Back Again

Some bacterial toxins and viruses have evolved the capacity to bind mammalian glycosphingolipids to gain access to the cell interior, where they can co-opt the endogenous mechanisms of cellular trafficking and protein translocation machinery to cause toxicity. Cholera toxin (CT) is one of the best-studied examples, and is the virulence factor responsible for massive secretory diarrhea seen in cholera. CT enters host cells by binding to monosialotetrahexosylganglioside (GM1 gangliosides) at the plasma membrane where it is transported retrograde through the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). In the ER, a portion of CT, the CT-A1 polypeptide, is unfolded and then “retro-translocated” to the cytosol by hijacking components of the ER associated degradation pathway (ERAD) for misfolded proteins. CT-A1 rapidly refolds in the cytosol, thus avoiding degradation by the proteasome and inducing toxicity. Here, we highlight recent advances in our understanding of how the bacterial AB5 toxins induce disease. We highlight the molecular mechanisms by which these toxins use glycosphingolipid to traffic within cells, with special attention to how the cell senses and sorts the lipid receptors. We also discuss several new studies that address the mechanisms of toxin unfolding in the ER and the mechanisms of CT A1-chain retro-translocation to the cytosol.

Some bacterial toxins and viruses have evolved the capacity to bind mammalian glycosphingolipids to gain access to the cell interior, where they can co-opt the endogenous mechanisms of cellular trafficking and protein translocation machinery to cause toxicity. Cholera toxin (CT) is one of the best-studied examples, and is the virulence factor responsible for massive secretory diarrhea seen in cholera. CT enters host cells by binding to monosialotetrahexosylganglioside (GM1 gangliosides) at the plasma membrane where it is transported retrograde through the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). In the ER, a portion of CT, the CT-A1 polypeptide, is unfolded and then “retro-translocated” to the cytosol by hijacking components of the ER associated degradation pathway (ERAD) for misfolded proteins. CT-A1 rapidly refolds in the cytosol, thus avoiding degradation by the proteasome and inducing toxicity. Here, we highlight recent advances in our understanding of how the bacterial AB5 toxins induce disease. We highlight the molecular mechanisms by which these toxins use glycosphingolipid to traffic within cells, with special attention to how the cell senses and sorts the lipid receptors. We also discuss several new studies that address the mechanisms of toxin unfolding in the ER and the mechanisms of CT A1-chain retro-translocation to the cytosol.

Insights on the trafficking and retro-translocation of glycosphingolipid-binding bacterial toxins

Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The two primary toxins, TcdA and TcdB, disrupt host cell function by inactivating small GTPases that regulate the actin cytoskeleton. This review will discuss the role of these two toxins in pathogenesis and the structural and molecular mechanisms by which they intoxicate cells. A focus will be placed on recent publications highlighting mechanistic similarities and differences between TcdA, TcdB, and different TcdB variants.

Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The two primary toxins, TcdA and TcdB, disrupt host cell function by inactivating small GTPases that regulate the actin cytoskeleton. This review will discuss the role of these two toxins in pathogenesis and the structural and molecular mechanisms by which they intoxicate cells. A focus will be placed on recent publications highlighting mechanistic similarities and differences between TcdA, TcdB, and different TcdB variants.

Toward a structural understanding of Clostridium difficile toxins A and B

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host–cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host–cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.