33 results on '"Pore-forming toxin"'
Search Results
2. Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- Author
-
Corkery, Dale P., Wu, Yao-Wen, Corkery, Dale P., and Wu, Yao-Wen
- Abstract
Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.
- Published
- 2023
- Full Text
- View/download PDF
3. Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- Author
-
Corkery, Dale P., Wu, Yao-Wen, Corkery, Dale P., and Wu, Yao-Wen
- Abstract
Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.
- Published
- 2023
- Full Text
- View/download PDF
4. Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- Author
-
Corkery, Dale P., Wu, Yao-Wen, Corkery, Dale P., and Wu, Yao-Wen
- Abstract
Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.
- Published
- 2023
- Full Text
- View/download PDF
5. Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- Author
-
Corkery, Dale P., Wu, Yao-Wen, Corkery, Dale P., and Wu, Yao-Wen
- Abstract
Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.
- Published
- 2023
- Full Text
- View/download PDF
6. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
- Author
-
Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, Wu, Yao-Wen, Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, and Wu, Yao-Wen
- Abstract
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
- Published
- 2022
- Full Text
- View/download PDF
7. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
- Author
-
Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, Wu, Yao-Wen, Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, and Wu, Yao-Wen
- Abstract
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
- Published
- 2022
- Full Text
- View/download PDF
8. Using structural biology to understand how bacterial toxins exert their activity
- Author
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Johnstone, Bronte Alexandria and Johnstone, Bronte Alexandria
- Abstract
A common mechanism to support bacterial survival and growth is the production of bacterial toxins. These may facilitate the infection of host cells by pathogenic bacteria or act on neighbouring bacteria to yield a competitive advantage. A particular mode of action, employed by a class of bacterial toxins known as pore-forming toxins (PFTs), is the formation of transmembrane pores in host cell membranes, most commonly causing cell lysis leading to cell death and release of nutrients. A major family of PFTs are the cholesterol-dependent cytolysins (CDCs). Secreted as water-soluble monomers, pore formation by CDCs is associated with substantial conformational changes resulting in the formation of a circular pore complex of around 35 – 40 monomers. More recently, we have identified a novel family of PFTs related to CDCs, but displaying significant differences in structure, mechanism, and biological function. The members of this novel family are referred to as the CDC-like (CDCL) proteins. A major focus of my PhD has been the investigation of CDCs and the novel CDCLs using multiple complimentary structural biology techniques, particularly X-ray crystallography, electron microscopy and small-angle X-ray scattering (SAXS). The first of the projects described in this thesis investigated how a conserved structural undecapeptide (UDP) in CDCs may play an allosteric role in pore-formation. Analysis of two different UDP mutants of the archetypal CDC perfringolysin O (PFO) suggest putative structural changes between these mutants compared to the wild-type protein. Further projects explored two novel CDCL members. The species Elizabethkingia anophelis produces two CDCLs referred to as ALY long (ALYL) and ALY short (ALYS) that possess three- and four-domain structures, respectively. The characterisation of the water-soluble form of these, including the determination of the crystal structure for ALYS, revealed structural similarities but also striking differences and novel structur
- Published
- 2022
9. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
- Author
-
Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, Wu, Yao-Wen, Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, and Wu, Yao-Wen
- Abstract
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
- Published
- 2022
- Full Text
- View/download PDF
10. Alciporin, a pore-forming protein as complementary defense mechanism in Millepora alcicornis
- Author
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Ministerio de Ciencia e Innovación (España), Caja Canarias, La Caixa, Gobierno de Canarias, Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (España), Ministerio de Sanidad, Consumo y Bienestar Social (España), Cabildo de Tenerife, Nocchi, Nathalia, González-Orive, Alejandro, Hernández-Creus, Alberto, Lorenzo-Morales, Jacob, Rodríguez, Adriana, Morchón, Rodrigo, Díaz-Marrero, Ana R., Fernández, José J., Ministerio de Ciencia e Innovación (España), Caja Canarias, La Caixa, Gobierno de Canarias, Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (España), Ministerio de Sanidad, Consumo y Bienestar Social (España), Cabildo de Tenerife, Nocchi, Nathalia, González-Orive, Alejandro, Hernández-Creus, Alberto, Lorenzo-Morales, Jacob, Rodríguez, Adriana, Morchón, Rodrigo, Díaz-Marrero, Ana R., and Fernández, José J.
- Abstract
Millepora alcicornis (Cnidaria: Hydrozoa), known as fire coral, is a tropical species settled in marine ecosystems of the Canary Islands in the last years. This hydrocoral biosynthesizes toxins involved in chemical defense and prey capture mechanisms. Toxicological studies have shown that the venom contained in the nematocysts of Millepora species is mainly composed of thermolabile proteins that display hemolytic activity, causing skin irritation and burn-like lesions upon contact. As a continuation of a previous study, the chromatographic fractionation of the aqueous extracts of M. alcicornis has confirmed the coexistence of proteins of different nature responsible for the hemolytic effects of red blood cells (RBCs) through two different mechanisms. Aside from the already described phospholipase A2 (PLA2) activity, in this work the presence of alciporin, a pore-forming protein (PFP), has been established for the first time for M. alcicornis. The sequence analysis revealed that alciporin fit an actinoporin with high homology to stichotoxins. The hemolytic effects of alciporin were analyzed and sphingomyelin was identified as its biological target. Also, the evolution of the hemolytic damage produced at the nanoscale has been studied using atomic force microscopy (AFM).
- Published
- 2022
11. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
- Author
-
Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, Wu, Yao-Wen, Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, and Wu, Yao-Wen
- Abstract
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
- Published
- 2022
- Full Text
- View/download PDF
12. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
- Author
-
Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, Wu, Yao-Wen, Jia, Xiaotong, Knyazeva, Anastasia, Zhang, Yu, Castro-Gonzalez, Sergio, Nakamura, Shuhei, Carlson, Lars-Anders, Yoshimori, Tamotsu, Corkery, Dale, and Wu, Yao-Wen
- Abstract
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
- Published
- 2022
- Full Text
- View/download PDF
13. Glabralysins, Potential New β-Pore-Forming Toxin Family Members from the Schistosomiasis Vector Snail Biomphalaria glabrata
- Author
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Lassalle, Damien, Tetreau, Guillaume, Pinaud, Silvain, Galinier, Richard, Crickmore, Neil, Gourbal, Benjamain, Duval, David, Lassalle, Damien, Tetreau, Guillaume, Pinaud, Silvain, Galinier, Richard, Crickmore, Neil, Gourbal, Benjamain, and Duval, David
- Abstract
Biomphalaria glabrata is a freshwater Planorbidae snail. In its environment, this mollusk faces numerous microorganisms or pathogens, and has developed sophisticated innate immune mechanisms to survive. The mechanisms of recognition are quite well understood in Biomphalaria glabrata, but immune effectors have been seldom described. In this study, we analyzed a new family of potential immune effectors and characterized five new genes that were named Glabralysins. The five Glabralysin genes showed different genomic structures and the high degree of amino acid identity between the Glabralysins, and the presence of the conserved ETX/MTX2 domain, support the hypothesis that they are pore-forming toxins. In addition, tertiary structure prediction confirms that they are structurally related to a subset of Cry toxins from Bacillus thuringiensis, including Cry23, Cry45, and Cry51. Finally, we investigated their gene expression profiles in snail tissues and demonstrated a mosaic transcription. We highlight the specificity in Glabralysin expression following immune stimulation with bacteria, yeast or trematode parasites. Interestingly, one Glabralysin was found to be expressed in immune-specialized hemocytes, and two others were induced following parasite exposure.
- Published
- 2020
- Full Text
- View/download PDF
14. An Inducible Cre-lox System to Analyze the Role of LLO in Listeria monocytogenes Pathogenesis.
- Author
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Nguyen, Brittney N, Nguyen, Brittney N, Portnoy, Daniel A, Nguyen, Brittney N, Nguyen, Brittney N, and Portnoy, Daniel A
- Abstract
Listeriolysin O (LLO) is a pore-forming cytolysin that allows Listeria monocytogenes to escape from phagocytic vacuoles and enter the host cell cytosol. LLO is expressed continuously during infection, but it has been a challenge to evaluate the importance of LLO secreted in the host cell cytosol because deletion of the gene encoding LLO (hly) prevents localization of L. monocytogenes to the cytosol. Here, we describe a L. monocytogenes strain (hlyfl) in which hly is flanked by loxP sites and Cre recombinase is under the transcriptional control of the L. monocytogenes actA promoter, which is highly induced in the host cell cytosol. In less than 2 h after infection of bone marrow-derived macrophages (BMMs), bacteria were 100% non-hemolytic. hlyfl grew intracellularly to levels 10-fold greater than wildtype L. monocytogenes and was less cytotoxic. In an intravenous mouse model, 90% of bacteria were non-hemolytic within three hours in the spleen and eight hours in the liver. The loss of LLO led to a 2-log virulence defect in the spleen and a 4-log virulence defect in the liver compared to WT L. monocytogenes. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of L. monocytogenes.
- Published
- 2020
15. An Inducible Cre-lox System to Analyze the Role of LLO in Listeria monocytogenes Pathogenesis.
- Author
-
Nguyen, Brittney N, Nguyen, Brittney N, Portnoy, Daniel A, Nguyen, Brittney N, Nguyen, Brittney N, and Portnoy, Daniel A
- Abstract
Listeriolysin O (LLO) is a pore-forming cytolysin that allows Listeria monocytogenes to escape from phagocytic vacuoles and enter the host cell cytosol. LLO is expressed continuously during infection, but it has been a challenge to evaluate the importance of LLO secreted in the host cell cytosol because deletion of the gene encoding LLO (hly) prevents localization of L. monocytogenes to the cytosol. Here, we describe a L. monocytogenes strain (hlyfl) in which hly is flanked by loxP sites and Cre recombinase is under the transcriptional control of the L. monocytogenesactA promoter, which is highly induced in the host cell cytosol. In less than 2 h after infection of bone marrow-derived macrophages (BMMs), bacteria were 100% non-hemolytic. hlyfl grew intracellularly to levels 10-fold greater than wildtype L. monocytogenes and was less cytotoxic. In an intravenous mouse model, 90% of bacteria were non-hemolytic within three hours in the spleen and eight hours in the liver. The loss of LLO led to a 2-log virulence defect in the spleen and a 4-log virulence defect in the liver compared to WT L. monocytogenes. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of L. monocytogenes.
- Published
- 2020
16. Contributions of the pore-forming toxin Listeriolysin O to Listeria monocytogenes pathogenesis
- Author
-
Nguyen, Brittney Nhu-Chau, Portnoy, Daniel A1, Nguyen, Brittney Nhu-Chau, Nguyen, Brittney Nhu-Chau, Portnoy, Daniel A1, and Nguyen, Brittney Nhu-Chau
- Abstract
Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Deletion of the gene encoding LLO, hly, or replacement of LLO with other CDCs results in strains that are 10,000-fold less virulent during mouse infections. Deletion of LLO also results in a strain that is immunosuppressive in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the PEST-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in myriad ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including MAPK activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address is whether LLO secreted in the cytosol has an impact on pathogenesis.To address whether LLO secreted in the cytosol impacts the pathogenesis of L. monocytogenes we engineered a strain, referred to as hlyfl, that deletes hly after escape from phagocytic vacuoles. Using hlyfl, we determined that LLO secreted in the cytosol causes cytotoxicity that impairs the growth of L. monocytogenes in macrophages. However, this strain was less virulent in mice than WT L. monocytogenes because it had a defect in cell-to-cell sprea
- Published
- 2019
17. Contributions of the pore-forming toxin Listeriolysin O to Listeria monocytogenes pathogenesis
- Author
-
Nguyen, Brittney Nhu-Chau, Portnoy, Daniel A1, Nguyen, Brittney Nhu-Chau, Nguyen, Brittney Nhu-Chau, Portnoy, Daniel A1, and Nguyen, Brittney Nhu-Chau
- Abstract
Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Deletion of the gene encoding LLO, hly, or replacement of LLO with other CDCs results in strains that are 10,000-fold less virulent during mouse infections. Deletion of LLO also results in a strain that is immunosuppressive in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the PEST-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in myriad ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including MAPK activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address is whether LLO secreted in the cytosol has an impact on pathogenesis.To address whether LLO secreted in the cytosol impacts the pathogenesis of L. monocytogenes we engineered a strain, referred to as hlyfl, that deletes hly after escape from phagocytic vacuoles. Using hlyfl, we determined that LLO secreted in the cytosol causes cytotoxicity that impairs the growth of L. monocytogenes in macrophages. However, this strain was less virulent in mice than WT L. monocytogenes because it had a defect in cell-to-cell sprea
- Published
- 2019
18. Roles of membrane vesicles in bacterial pathogenesis
- Author
-
Vdovikova, Svitlana and Vdovikova, Svitlana
- Abstract
The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the
- Published
- 2017
19. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death
- Author
-
Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, Wai, Sun N., Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, and Wai, Sun N.
- Abstract
Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.
- Published
- 2017
- Full Text
- View/download PDF
20. Roles of membrane vesicles in bacterial pathogenesis
- Author
-
Vdovikova, Svitlana and Vdovikova, Svitlana
- Abstract
The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the
- Published
- 2017
21. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death
- Author
-
Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, Wai, Sun N., Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, and Wai, Sun N.
- Abstract
Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.
- Published
- 2017
- Full Text
- View/download PDF
22. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death
- Author
-
Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, Wai, Sun N., Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, and Wai, Sun N.
- Abstract
Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.
- Published
- 2017
- Full Text
- View/download PDF
23. Roles of membrane vesicles in bacterial pathogenesis
- Author
-
Vdovikova, Svitlana and Vdovikova, Svitlana
- Abstract
The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the
- Published
- 2017
24. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death
- Author
-
Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, Wai, Sun N., Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, and Wai, Sun N.
- Abstract
Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.
- Published
- 2017
- Full Text
- View/download PDF
25. Roles of membrane vesicles in bacterial pathogenesis
- Author
-
Vdovikova, Svitlana and Vdovikova, Svitlana
- Abstract
The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the
- Published
- 2017
26. Roles of membrane vesicles in bacterial pathogenesis
- Author
-
Vdovikova, Svitlana and Vdovikova, Svitlana
- Abstract
The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the
- Published
- 2017
27. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death
- Author
-
Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, Wai, Sun N., Vdovikova, Svitlana, Luhr, Morten, Szalai, Paula, Skalman, Lars Nygård, Francis, Monika K., Lundmark, Richard, Engedal, Nikolai, Johansson, Jörgen, and Wai, Sun N.
- Abstract
Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.
- Published
- 2017
- Full Text
- View/download PDF
28. Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity.
- Author
-
Gómez, Isabel, Gómez, Isabel, Sánchez, Jorge, Muñoz-Garay, Carlos, Matus, Violeta, Gill, Sarjeet S, Soberón, Mario, Bravo, Alejandra, Gómez, Isabel, Gómez, Isabel, Sánchez, Jorge, Muñoz-Garay, Carlos, Matus, Violeta, Gill, Sarjeet S, Soberón, Mario, and Bravo, Alejandra
- Abstract
Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.
- Published
- 2014
29. Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity.
- Author
-
Gómez, Isabel, Gómez, Isabel, Sánchez, Jorge, Muñoz-Garay, Carlos, Matus, Violeta, Gill, Sarjeet S, Soberón, Mario, Bravo, Alejandra, Gómez, Isabel, Gómez, Isabel, Sánchez, Jorge, Muñoz-Garay, Carlos, Matus, Violeta, Gill, Sarjeet S, Soberón, Mario, and Bravo, Alejandra
- Abstract
Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.
- Published
- 2014
30. Molecular Modeling of Bacterial Nanomachineries
- Author
-
Dal Peraro, Matteo, Degiacomi, Matteo Thomas, Dal Peraro, Matteo, and Degiacomi, Matteo Thomas
- Abstract
Proteins have the ability to assemble in multimeric states to perform their specific biological function. Unfortunately, characterizing experimentally these structures at atomistic resolution is usually difficult. For this reason, in silico methodologies aiming at predicting how multiple protein copies arrange to forma multimeric complex would be desirable. We present Parallel OptimizationWorkbench (POW), a swarm intelligence based optimization framework able to deal, in principle, with any optimization problem. We show that POW can be applied to biologically relevant problems such as prediction of protein assemblies and the parameterization of a Coarse-Grained force field for proteins. By combining POW optimizations, Molecular Dynamics simulations, Poisson-Boltzmann calculations and a variety of experiments, we subsequently study two bacterial nanomachieries: Aeromonas hydrophila's pore-forming toxin aerolysin, and Yersinia enterocolitica injectisome. These structures are challenging both for their size, and for the timescales involved in their functioning. Aerolysin is a pore-forming toxin secreted as an hydrophilic monomer. By means of large conformational changes, the protein heptamerizes on the target cell's surface, and finally inserts β-barrel into its lipid bilayer, causing cell death. The main hurdle in the study of this structure is the complexity of the mode of action, which spans timescales currently unreachable by classical molecular dynamics. We show that aerolysin C-terminal region has the dual role of preventing premature oligomerization and helping the folding of tertiary structure, qualifying therefore as an intramolecular chaperone. We study the transmembrane β-barrel properties and compare them with those of the homologous protein α-hemolysin. We show that aerolysin's barrel is more rigid than α-hemolysin's, and should be anion selective. We present models for aerolysin heptamer both in prepore and, for the first time, in membrane-inserted confor
- Published
- 2012
- Full Text
- View/download PDF
31. Plasma Membrane-porating Domain in Poliovirus 2B Protein. A Short Peptide Mimics Viroporin Activity
- Author
-
Dirección General de Investigación Científica y Técnica, DGICT (España), Eusko Jaurlaritza, Universidad del País Vasco, Fundación Ramón Areces, Università degli Studi di Ferrara, Madan, Vanesa, Sánchez-Martínez, Silvia, Vedovato, Natascia, Rispoli, Giorgio, Carrasco Llamas, Luis, Nieva, José Luis, Dirección General de Investigación Científica y Técnica, DGICT (España), Eusko Jaurlaritza, Universidad del País Vasco, Fundación Ramón Areces, Università degli Studi di Ferrara, Madan, Vanesa, Sánchez-Martínez, Silvia, Vedovato, Natascia, Rispoli, Giorgio, Carrasco Llamas, Luis, and Nieva, José Luis
- Abstract
Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35–55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC50 ≈ 4 × 10−7 M) and boundary liposome bilayers (starting at peptide to lipid molar ratios > 1:104). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10−8 M. Thus, we have established a “one-helix” 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection
- Published
- 2007
32. Outer membrane vesicle-mediated export of a pore-forming cytotoxin from Escherichia coli
- Author
-
Kouokam, J Clavin, Wai, Sun Nyunt, Kouokam, J Clavin, and Wai, Sun Nyunt
- Abstract
ClyA, also called SheA or HlyE, is a four-helix bundle cytotoxic protein expressed by Escherichia coli and other enterobacteria. The expression of ClyA was shown to be controlled by the nucleoid protein H-NS and could be activated by overproduction of several different transcriptional regulators such as SlyA, MprA, HlyX, and FnrP. The ClyA protein contains two hydrophobic potential transmembrane domains. Lipid bilayer experiments and electron microscopic studies have led to the conclusion that ClyA forms stable pores in target membranes by assembling into ring-shaped toxin oligomers, but little or no effect was found on the bacterial cell membranes from which it is produced, presumably because the lytic activity of the protein is stimulated by cholesterol. Several studies have revealed that the ClyA toxin, which does not have any canonical signal sequence, nevertheless is secreted to the medium. It has become evident that a vesicle-mediated transport mechanism is responsible for the activation and delivery of ClyA protein, seemingly independent of the previously described type I-V secretion systems.
- Published
- 2006
- Full Text
- View/download PDF
33. Molecular Genetic Approaches to Biomolecular Materials
- Author
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TEXAS A AND M UNIV COLLEGE STATION HEALTH SCIENCE CENTER, Bayley, Hagan, TEXAS A AND M UNIV COLLEGE STATION HEALTH SCIENCE CENTER, and Bayley, Hagan
- Abstract
The leukocidins are bicomponent pore-forming toxins that are structurally related to staphylococcal alpha-hemolysin, a single component toxin. alpha-Hemolysin has many applications in the area of biomolecular materials. Therefore, leukocidins have now been characterized to expand the set of tools available for materials engineering. The approach taken in the AASERT project was to obtain genes for the two components of the toxin, to express them in E. coli, to characterize the heterologously expressed pores by bilayer recording, and to use molecular genetic technology to explore structure- function relationships in these proteins. The first three of the four objectives have been accomplished.
- Published
- 2000
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