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Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle). Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.

Intraprostatic injection of BTX-A has demonstrated clinical improvement in men with bladder out let obstruction. Firstly, we investigated the mechanisms of action of BTX-A on the prostate. Secondly, an animal model for nonbacterial prostatitis in rats was developed using intraprostatic injection of capsaicin, an agent thought to excite c-afferent fibers and cause neurogenic inflammation. The analgesic and anti-inflammatory properties of Botulinum toxin type A (BoNT-A) was tested in this model. (1) Adult male Spragu-Dawley rats were injected with varying doses of BTX-A into the prostate, and the prostates were harvested after 1 or 2 weeks. The effects of BTX-A on prostate histology, and the proliferative and apoptotic indexes were determined using hematoxylin and eosin staining, proliferative cell nuclear antigen staining and TUNEL staining, respectively. Changes in a1A adrenergic receptor and androgen receptor were evaluated by Western blotting. (2) Adult male Spragu-Dawley rats were injected with varying doses of capsaicin into the prostate. The nociceptive effects of capsaicin were evaluated for 30 min by using a behavior approach; the prostate was removed for histology and cyclooxygenase (COX) 2 concentration measurement. Evans blue (50mg/kg) was also injected intravenously to assess for plasma protein extravasation. The other set of animals were injected with up to 20U of BoNT-A into the prostates 1 wk prior to intraprostatic injection of 1000umol/l capsaicin. (1) One week after BTX-A injection generalized prostate atrophy was observed. There was a significant increase in apoptotic cells (12, 16 and 22-fold), and decrease in proliferative cells (38%, 77% and 80%) and a1A adrenergic receptor (13%, 80% and 81%) for 5U, 10U and 20U, respectively. There was no significant change in androgen receptors. The effects were decreased 2 weeks after BTX-A treatment. (2) Capsaicin dose dependently induced modifications in pain behavior closing of the eyes, hypolocomotion, and inflammatory changes: increase of inflammatory cell accumulation, COX2 expression, and plasma extravasation at the acute stage, but completely recovered at 1 wk. BoNT-A pretreatment dose dependently reversed pain behavior and inflammation. BoNT-A 20U significantly decreased inflammatory cell accumulation, COX2 expression, and Evans blue extraction (82.1%, 93.0% and 50.4, respectively), and reduced pain behavior (66.% for eye score and 46.5% for locomotion score). Conclusion (1): BTX-A injection into the prostate alters cellular dynamics by inducing apoptosis, inhibiting proliferation and down-regulating a1A adrenergic receptors. BTX-A may potentially be the drug that has dual actions on the static and dynamic components of benign prostatic hyperplasia. (2): Intraprostatic capsaicin injection induced neurogenic prostatitis and prostatic pain, and may be a useful research model. BoNT-A produced anti-inflammatory and analgesic effects, and support clinical evaluation in prostatitis.

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.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

Perfringolysin O (PFO), a cytolytic toxin from by the pathogenic bacterium Clostridium perfringens, perforates mammalian cell membranes by forming large aqueous pores. Secreted as water-soluble monomers, the toxin molecules bind to cholesterol-containing membranes, form large, circular oligomeric complexes on the membrane surface and then insert into the bilayer to create pores with diameters near 300 à . Using multiple independent fluorescence techniques as primary tools, the mechanisms of PFO membrane binding and oligomerization have been identified. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. Remarkably, only the short hydrophobic loops at the tip of the D4 β-sandwich are exposed to the bilayer interior, while the remainder of D4 projects from the membrane surface. Thus, a very limited interaction of D4 with the bilayer core appears to be sufficient to accomplish cholesterol recognition and initial PFO binding to the membrane. Upon PFO membrane binding, a structural element in domain 3 (D3) of the molecule moves to expose the edge of a previously-hidden β-strand that forms the monomer-monomer interface. The β-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack to align the transmembrane β-hairpins of adjacent monomers in the proper register for insertion. Membrane-dependent structural rearrangements are thus required to initiate and regulate PFO oligomerization. Fluorescence resonance energy transfer measurements reveal that the elongated toxin monomer arrives at the membrane in an ‘end-on’ orientation, with its long axis oriented nearly perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form a prepore complex. In particular, the D3 polypeptide segments that form the transmembrane β-hairpins remain far above the membrane surface both at the membrane-bound monomer and prepore stages of pore formation. However, upon pore formation, the height of the oligomeric complex above the membrane surface is significantly reduced. The major topographical changes that occur during the prepore-to-pore transition of the PFO oligomer, therefore appears to result primarily from a collapsing of the extended domain 2 (D2) conformation in the monomer.

Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Timothy M. Logan, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Oct. 2, 2003). Includes bibliographical references.

This thesis is an investigation of the binary toxin of Clostridium difficile. The aim was to improve the understanding of the role of the binary toxin in pathogenesis. Quantitative real-time PCR (qPCR) was used to study expression of cdtA encoding the binary toxin enzymatic domain, cdtR encoding the binary toxin regulator and tcdB encoding toxin B, in response to growth phase and antimicrobial treatments in 2 C. difficile strains. Validation of a set of stable reference genes was required prior to qPCR analysis of gene expression. A universal set of genes could not be identified and reference genes should be validated on a strain-specific basis. Significant increases or decreases in expression were observed in response to levofloxacin and enrofloxacin exposure. The 2 strains selected were from different ribotypes and did not always share expression patterns. Binary toxin loci were sequenced in and compared between 10 C. difficile strains. A non-sense mutation in the cdtR gene of a ribotype 078 strain was identified and found to be restricted to toxinotype V isolates. This mutation is predicted to result in a truncated, non-functional protein. Despite the mutation, cdtA expression was still detected by qPCR. Finally, an evaluation of commercial nucleic acid extraction kits was performed. All kits produced RNA of adequate quality and yield, however, RNA isolated using the the Roche MagNA Pure LC RNA Isolation Kit could not be analyzed using the Agilent Bioanalyzer. It could not properly assign RNA integrity numbers due to a failure to remove small RNAs which were interpreted as degradation. All kits were suitable for DNA extraction with the exception of the MagNA Pure LC DNA Isolation Kit III which produced sheared DNA. In conclusion, this study demonstrated that the binary toxin regulator isn’t necessary for toxin expression and suggests other regulators of expression exist. Binary toxin gene expression did not necessarily correlate with expression of tcdB and expression levels vary between strains. This study also highlighted how the heterogeneity of C. difficile complicates gene expression experiments and the need for assessment of nucleic acid extraction methods due to critical variations between established commercial systems.