CAMP factor is an extracellular pore-forming toxin secreted by the group B streptococci Streptococcus agalactiae. In conjunction with the action of sphingomyelinase secreted by Staphylococcus aureus, which converts membrane sphingomyline to ceramide, CAMP factor kills susceptible cells by creating holes in them. Since the monomeric or oligomeric structure of CAMP factor is not yet known, no studies on the membrane-penetrating domain of this toxin have been done. In the present study, the interaction of a putative hydrophobic domain between residues T90 and V115 with the target membrane was examined by cysteine-scanning mutagenesis and site-selective fluorescent labeling. The combination of steady state and lifetime fluorescence measurements and collisional quenching experiments with nitroxide labeled fatty acids indicate that residues from T90 to V115 contact the membrane upon binding and oligomerization of CAMP factor on cell membranes. More importantly, all these individual assays indicate that the residues from N104C to F109C insert superficially into the membrane with a β-sheet conformation.

CAMP factor is an extracellular pore-forming toxin secreted by the group B streptococci Streptococcus agalactiae. In conjunction with the action of sphingomyelinase secreted by Staphylococcus aureus, which converts membrane sphingomyline to ceramide, CAMP factor kills susceptible cells by creating holes in them. Since the monomeric or oligomeric structure of CAMP factor is not yet known, no studies on the membrane-penetrating domain of this toxin have been done. In the present study, the interaction of a putative hydrophobic domain between residues T90 and V115 with the target membrane was examined by cysteine-scanning mutagenesis and site-selective fluorescent labeling. The combination of steady state and lifetime fluorescence measurements and collisional quenching experiments with nitroxide labeled fatty acids indicate that residues from T90 to V115 contact the membrane upon binding and oligomerization of CAMP factor on cell membranes. More importantly, all these individual assays indicate that the residues from N104C to F109C insert superficially into the membrane with a β-sheet conformation.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

The discovery of the pore-forming toxins produced by Bacillus thuringiensis, which are toxic to insects but not to mammalians, has provided a new successful means to control harmful plant-feeding insects biologically. The toxins are also used on insects that don’t feed on plants, for example on Anopheles. The Bacillus thuringiensis toxins fall into two structural families, named cry and cyt. All of these toxins act by damaging the cell membranes in the mid gut of the insect. In this study, a reliable system for expression and purification of the recombinant Cyt2Aa1 toxin has been developed. The recombinant Cyt2Aa1 toxin has been produced, characterized, followed by the construction of the cysteine mutants V186C and L189C by site directed mutagenesis. The new expression system yields 0.4 g of protein per litre of culture. The activated Cyt2Aa1 toxin is active in the hemolysis assay. Of note, the hemolytic activity of the V186C mutant exceeds that of wild type Cyt2Aa1 toxin and of the L189C mutant. Calcein release assay experiments have been done to examine the activity of the toxin with different artificial liposomes. It was found that Cyt2Aa1 toxin is very active with DMPC, DMPC+DMPG unilamellar liposomes. Surprisingly, however, Cyt2Aa1 toxin showed no activity with liposomes containing cholesterol. With both erythrocytes and sensitive liposomes, the toxin shows a “pro-zone effect”, that is the activity decreases at very high concentrations. The findings are discussed in the context of the toxin’s putative mode of action.

The discovery of the pore-forming toxins produced by Bacillus thuringiensis, which are toxic to insects but not to mammalians, has provided a new successful means to control harmful plant-feeding insects biologically. The toxins are also used on insects that don’t feed on plants, for example on Anopheles. The Bacillus thuringiensis toxins fall into two structural families, named cry and cyt. All of these toxins act by damaging the cell membranes in the mid gut of the insect. In this study, a reliable system for expression and purification of the recombinant Cyt2Aa1 toxin has been developed. The recombinant Cyt2Aa1 toxin has been produced, characterized, followed by the construction of the cysteine mutants V186C and L189C by site directed mutagenesis. The new expression system yields 0.4 g of protein per litre of culture. The activated Cyt2Aa1 toxin is active in the hemolysis assay. Of note, the hemolytic activity of the V186C mutant exceeds that of wild type Cyt2Aa1 toxin and of the L189C mutant. Calcein release assay experiments have been done to examine the activity of the toxin with different artificial liposomes. It was found that Cyt2Aa1 toxin is very active with DMPC, DMPC+DMPG unilamellar liposomes. Surprisingly, however, Cyt2Aa1 toxin showed no activity with liposomes containing cholesterol. With both erythrocytes and sensitive liposomes, the toxin shows a “pro-zone effect”, that is the activity decreases at very high concentrations. The findings are discussed in the context of the toxin’s putative mode of action.

No description available.

Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can be only activated when exposed to specific cell-surface proteases produced by a specific cell type. We ultimately want to target breast cancer cells that differentially secrete or express proteases such as matrix-metalloproteases (Stautz D et al., 2012; Zhang, M et al. 2013). To engineer this protein we took advantage of the N-terminus of ClyA. The N-terminus of ClyA, which is highly hydrophobic (Oscarsson J et al), undergoes a conformational change to insert into the target cell membrane (Oscarsson J et al). This conformational change allows ClyA to penetrate the target membrane to form a transmembrane domain of ClyA. The hydrophobic nature of lipid membranes makes it highly unfavorable for any charged residues to cross the membrane (Hunt J 1997). With this in mind, we hypothesize that negative charges inserted into the N-terminus of ClyA will inhibit it from inserting into the membrane. Thus, we mutated the N-terminus of the ClyA protein by inserting an inactivation site composed of negatively charged amino acids that we hypothesize would prevent insertion into the plasma membrane of the target cell. Once we confirmed that this construct was an inactive ClyA mutant, we inserted a thrombin cleavage site right after the inserted negative charges. This site should allow us to remove the negative charges once the protein is exposed to thrombin. Once the negative charges are removed, the protein should recover its activity. This approach will allow us to create a version of ClyA that is protease-switchable.

Porous ceramics have successfully been used in a wide variety of highly advanced applications. Current routes to porous ceramics are limited in the types of porosity they can create and no one process is flexible enough to create any desired structure. This study introduces the use of ice particles as pore forming agents to fabricate porous materials. This novel method possesses several advantages over current industrial techniques including environmental friendliness, low cost, and flexibility in size and shape of resulting pores. Porous ceramic structures were created by adding preformed ice particles to an alumina slurry which was quickly frozen, air dried, and then sintered. Porosity was characterized using Scanning Electron Microscopy (SEM), Archimedes measurements, and gas sorption techniques. Small spherical pores were successfully created in the 20-200?m range and larger spherical pores were also created in the 2-3 mm range. Amount of porosity was controlled through specifying the amount of ice added to the ceramic slurry. Samples were prepared with porosity levels ranging from 30-75%. As a completely new process, these initial results are quite promising and further development will allow for even greater morphology control. / Master of Science

Pseudomonas aeruginosa est un pathogène opportuniste responsable de maladies nosocomiales. Il provoque des infections aiguës ou chroniques en employant conjointement plusieurs facteurs de virulence. Les souches les plus agressives possèdent un système de sécrétion de type III (SST3), injectant des toxines directement dans le cytoplasme des cellules eucaryotes grâce à une nano-aiguille. Récemment, une souche clinique hyper-virulente, appelée CLJ1, a été isolée dans l'unité de soins intensifs de l'hôpital universitaire de Grenoble sur un patient souffrant d'une infection pulmonaire hémorragique. Cette souche ne possède pas les gènes codant pour le SST3 mais sécrète une pore-forming toxin, ExlA, non identifiée auparavant. ExlA est une protéine de 172 kDa, formant des pores de 1,6 nm dans la membrane plasmique de plusieurs types de cellules, à l'exception des érythrocytes. Le pore provoque la rétraction des cellules hôtes et finit par induire la mort de la cellule. Nous avons montré que CLJ1 appartenait à un nouveau clade très divergent des souches classiques de P. aeruginosa, dont les membres possèdent le gène exlA au lieu des gènes codant pour le SST3. Les souches exlA-positives que nous avons collectées dans le monde proviennent d'infections humaines et d'échantillons environnementaux. Leur cytotoxicité, sur diverses cellules humaines et sur un modèle murin d’infection pulmonaire, est corrélée avec les niveaux de sécrétion d'ExlA. En plus de la toxicité membranaire, les souches exlA-positives ont montré des activités protéolytiques élevées envers les VE et E-cadhérines, deux protéines adhésives des jonctions adhérentes requises pour l'intégrité de l'endothélium et de l'épithélium, respectivement. Nous avons démontré que la formation de pores par ExlA dans la membrane eucaryote induisait une entrée massive et rapide de calcium dans le cytosol. Cet afflux de calcium permet la maturation et l'activation d'ADAM10, une protéase eucaryote située à la membrane plasmique. L'activation d’ADAM10 induit le clivage de ses substrats naturels : les VE et E-cadhérines. ExlA fait partie de la même famille de pore forming toxin que ShlA de Serratia marcescens. Nous avons démontré que ShlA utilisait le même mécanisme qu’ExlA pour induire le clivage des cadhérines. En conclusion, les souches bactériennes produisant ExlA ou ShlA détournent un mécanisme naturel de l'hôte pour induire la perte d'intégrité tissulaire. / Pseudomonas aeruginosa is an opportunistic pathogen responsible for nosocomial diseases. It provokes acute or chronic infections due to several virulence factors acting in concert. The most aggressive strains possess a Type III Secretion System (T3SS), injecting toxins directly into the cytoplasm of eukaryotic cells thanks to a nano-needle. Recently, a hyper-virulent clinical strain, called CLJ1, was isolated from a patient suffering of hemorrhagic pulmonary infection, at the intensive care unit of Grenoble University Hospital. This strain lacks a T3SS but secretes a pore-forming toxin, ExlA, not previously identified. ExlA is a 172-kDa protein, forming 1.6-nm pores in the plasma membrane of several cell types, except erythrocytes. The pore causes the retraction of host cells and eventually induces necrotic cell death. We showed that CLJ1 belongs to a recently-discovered and highly divergent clade of P. aeruginosa, whose members possess the exlA gene instead of the genes coding for the T3SS and its effectors. The strains we collected worldwide originate from human infections and environmental samples. Their cytotoxicity on various human cells and mouse models of infection was correlated with ExlA secretion levels. In addition to membrane toxicity, exlA-positive strains displayed high proteolytic activities targeting VE and E-cadherins, two intercellular-junction adhesive proteins required for endothelium and epithelium integrity. We thus investigated the mechanisms of ExlA-induced cadherin cleavage. We demonstrated that ExlA pore formation in the eukaryotic membrane induces a massive and rapid entry of calcium into the cytosol. This calcium influx enables the maturation and activation of ADAM10, an eukaryotic protease located at the cell membrane. ADAM10 activation induces the cleavage of its natural substrates: the VE- and E-cadherins. ExlA is related to other toxins, including ShlA from Serratia marcescens, and altogether they constitute a family of pore-forming toxins with unique properties. We demonstrated that ShlA uses the same mechanism as ExlA to induce the cleavage of the cadherins. In conclusion, exlA- and shlA-positive strains hijack a natural mechanism of the host to induce the loss of tissue integrity.