Venoms of the Sicariidae spider family contain phospholipase D (PLD) enzyme toxins that can cause severe dermonecrosis and even death in humans. PLD toxins are known to cleave the substrates sphingomyelin (SM) and lysophosphatidylcholine (LPC) in mammalian tissues, releasing a choline headgroup and a reported monoester phospholipid formed via a hydrolytic reaction. However, some PLD toxins have demonstrated the ability to utilize substrates besides SM and LPC and other PLD toxins have demonstrated no activity against either SM or LPC. Given that the etiology of the disease state following envenomation is not well understood, we postulated that PLD toxins could be utilizing other phospholipid substrates in vivo. To determine the level of promiscuity among the PLD toxins, we developed a novel ³¹P-NMR assay to measure phospholipase activity against a panel of potential phospholipid substrates. While developing the assay, we made the surprising discovery that recombinant PLD toxins, as well as whole venoms from diverse Sicariidae species, exclusively generates cyclic phosphate rather than hydrolytic products. We also found that a distantly related PLD toxin from a pathogenic bacterium, with low sequence identity to the spider PLDs, exclusively generates cyclic phosphate products. We then established that St_βIB1i, a PLD with extremely diminished activity toward SM and LPC, actually demonstrates large preferential specificity towards ethanolamine phospholipid substrates. We solved the crystal structure of St_βIB1i to compare to PLD toxins of known structure, toward an understanding of the molecular basis of substrate specificity. The cyclic phosphate products generated by the PLD toxins have extremely different biochemical properties than their monoester counterparts and may be relevant to the pathology following envenomation or bacterial infection. In addition the specificity St_βIB1i has for ethanolamine substrates may have biological implications, as insects have high concentrations of ethanolamine-containing phospholipids.

Die Struktur der Plasmamembran ist von deren Lipid- und Proteinzusammensetzung abhängig und wird durch die Anbindung an das unterliegende Zytoskelett beeinflusst. Das Ziel der vorliegenden Arbeit war die Untersuchung eines neuen Modellsystems basierend auf po­ren­über­span­nen­den Membranen, welches sowohl die heterogene Lipidzusammensetzung als auch den Einfluss eines unterliegenden Netzwerks berücksichtigt. Lipidmembranen, zusammengesetzt aus der „raft“-ähnlichen Lipidmischung DOPC/Sphingo­myelin/Cho­les­terin (40:40:20), wurden auf porösen, hochgeordneten Siliziumsubstraten mit Po­ren­durch­messern von 0.8, 1.2 und 2.0 µm durch Spreiten und Fusion von Riesenvesikeln (giant unilamellar vesicles, GUVs) präpariert. Die mikroskopische Phasenseparation in koexistierenden flüssig-geordneten (liquid ordered, lo) und flüssig-ungeordneten (liquid disordered, ld) Domänen wurde stark durch das unterliegende poröse Substrat beeinflusst. Die Größe der lo-Domänen konnte durch die Porengröße des Siliziumsubstrats, die Temperatur und den Cholesteringehalt der Membran, welcher durch Zugabe von Methyl-β-Cyclodextrin moduliert wurde, kontrolliert werden. Die Bindung der Shiga Toxin B-Untereinheit (STxB) an po­ren­überspannende Membranen, dotiert mit 5 mol% des Rezeptorlipids Gb3, führte zu einem Anstieg des Anteils der lo-Phase. Außerdem wurde die Bildung von lo-Domänen in nicht-phasenseparierten Membranen, zusammengesetzt aus DOPC/Sphingomyelin/Cholesterin/Gb3 (65:10:20:5), durch die Shiga Toxin-Bindung induziert. Ein Anstieg des Anteils der lo-Phase konnte ebenfalls bei der Bindung der pentameren Cholera Toxin B-Untereinheit (CTxB) an po­ren­überspannende Membranen, dotiert mit 1 mol% des Rezeptorlipids GM1, beobachtet werden. Des Weiteren wurde der Einfluss der chemischen Struktur des Gb3-Moleküls auf die Shiga Toxin-Bindung und die Reorganisation von festkörperunterstützten Membranen (solid supported membranes, SSMs) untersucht. Die STxB-Bindung an α-hydroxyliertes Gb3 erhöhte signifikant den Anteil der lo-Phase, während eine cis-Doppelbindung zur Bildung einer weiteren lo-Phase führte, die vermutlich ungesättigte (Glyko-)Sphingolipide und Cholesterin enthält. Im Falles des ungesättigten Gb3 konnte außerdem eine Kondensation zu größeren Domänen nach der STxB-Bindung beobachtet werden. Die genaue Phasenzuordnung der eingesetzten Glykospingolipide vor der Proteinbindung ist bisher unbekannt. Daher wurde das Phasenverhalten eines fluoreszierenden Polyen-Ga­lac­to­ce­re­bro­sids untersucht, welches bevorzugt in der lo-Phase von GUVs angereichert war. Dieser neue, intrinsische Fluorophor vermag als Grundlage für weitere Studien zum Phasenverhalten von Glykosphingolipiden dienen.

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.

This study sought to understand the pathogenesis of hemorrhagic abomasitis in calves by characterizing a type A Clostridium perfringens isolate. The complete genome sequence of an isolate from an outbreak of hemorrhagic abomasitis was compared to the three complete C. perfringens genomes currently available in GenBank. Unique findings included the presence of an integrated plasmid sequence and a frameshift mutation in the virS gene, which encodes the main sensor kinase that controls virulence gene regulation. An ~ 55 kb plasmid similar to pCW3 was found, in addition to two smaller plasmids with no significant similarity to available C. perfringens plasmid sequences. A number of plasmid-related fragments were also identified. Neither genomic nor proteomic approaches identified novel toxins, but an alternate and unexpected picture of virulence has emerged suggesting that anomalous virulence gene regulation might contribute to pathogenicity in this isolate. / Natural Sciences and Engineering Research Council, the Ontario Graduate Scholarship, the Animal Health Strategic Initiative of the Ontario Ministry of Agriculture, Food and Rural Affairs

The main objective of this work was to develop an antigen-capture enzyme-linked immunosorbent assay for detection of beta2-toxin in the intestine of neonatal piglets. The format of the assay comprised of capture antibodies (polyclonal), antigen (beta2-toxin), detecting antibody (labeled monoclonal) and a substrate. The ELISA was optimized using recombinant protein. After intestinal content samples were applied, the test protocol needed to be adjusted because of the presence of high background signal in some samples consistent with intestinal proteases. This was overcome by processing the samples at 4oC and using citrate buffer pH 6.1 containing 5% bovine serum albumin. The second objective was to identify cpb2 in Clostridium perfringens type A isolates from selected animal species and to examine genotype-phenotype corelation. The study concluded that consensus cpb2, if present, was almost always expressed. In contrast, only about three-quarters of atypical cpb2, mostly was present in isolates of non-porcine origin, were expressed. / Ontario Ministry of Agriculture, Food and Rural Affairs; The Natural Sciences and Engineering Research Council of Canada

Chromosomal toxin-antitoxin (TA) modules have been proposed to function as regulators of cell growth in response to environmental perturbations. The objective of this study was to characterize the MazEF TA system of the human pathogen Streptococcus mutans. Our data showed that the mazEF genes form a bicistronic operon. MazF toxin had a toxic effect on cells and this effect can be neutralized by coexpression of its cognate antitoxin MazE. Furthermore, we demonstrated that MazE and MazF proteins interact with each other in vivo, confirming the nature of this TA as a type II addiction system. We also demonstrated that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a protein complex. Our results suggest that the MazEF TA might represent a cell growth modulator facilitating the persistence of S. mutans in the oral cavity.

Chromosomal toxin-antitoxin (TA) modules have been proposed to function as regulators of cell growth in response to environmental perturbations. The objective of this study was to characterize the MazEF TA system of the human pathogen Streptococcus mutans. Our data showed that the mazEF genes form a bicistronic operon. MazF toxin had a toxic effect on cells and this effect can be neutralized by coexpression of its cognate antitoxin MazE. Furthermore, we demonstrated that MazE and MazF proteins interact with each other in vivo, confirming the nature of this TA as a type II addiction system. We also demonstrated that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a protein complex. Our results suggest that the MazEF TA might represent a cell growth modulator facilitating the persistence of S. mutans in the oral cavity.

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.

Recently a whole cell based biosensor has been developed in our laboratory that consists of a monolayer of human umbilical vein endothelial cells (HUVECs) on the asymmetric cellulose triacetate (CTA) membrane of an ion selective electrode (ISE). When a confluent cell monolayer is formed across the membrane, response from the sensor is inhibited due to inhibited ion transport across the membrane. When the cell based biosensor is exposed to permeability modifying agents, the permeability across the cell monolayer is altered facilitating more ion transport and as a result the response from the sensor increases. This sensor response can be related to the concentration of these agents. One objective of this research was to investigate the ability of the sensor to detect a physiological toxin, alpha toxin from Staphylococcus aureus. Studies demonstrated that the biosensor can detect 0.1ng/ml alpha toxin. Considering the fact that the concentration of this toxin is 100-250 ng/ml in whole blood in humans, this biosensor has the ability to act as the diagnostic tool for staphylococcal diseases. Another part of this research was to investigate the ability of the biosensor to measure angiogenesis by measuring the changes in permeability induced by cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and tumor necrosis factor andamp;aacute; (TNF- andamp;aacute;) individually and in combination. The sensor response was then compared with the common in vitro assays for angiogenesis. The study demonstrated that at the concentrations studied the sensor response in the presence of cytokines was much higher than that observed for other angiogenesis assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis. Furthermore, epithelial cell based sensor responses to the same cytokines were compared with the responses from endothelial cell based sensor and the mechanisms behind the increased sensor response were elucidated. Finally, to investigate the ability of the sensor to screen cancer, the biosensor was exposed to the serum from healthy individuals and cancer patients. The results showed that the sensor can distinguish between healthy individuals and cancer patients and the results correlate with the stages of cancer.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.