Many bacterial toxins are important virulence factors, capable of instigating marked changes in the physiology of susceptible cells and tissues. Mechanisms of membrane attack by Vibrio toxins were examined on target cells, using cell physiological techniques, in particular, assays of haemolysis and radioisotope movement. Kanagawa haemolysin (KH; commercially available preparation of the thermostable direct haemolysin (TDH)) of V. parahaemolyticus caused lysis of human (but not horse or hagfish) erythrocytes that occurred (a) after colloid osmosis due to raised cation permeability, (b) independently of the KH:red cell ratio, and (c) with a monovalent cation selectivity series (reversed Eisenman VIII with a small K+ anomaly). The binding phase of KH was longer than the 1 - 2 minutes suggested by other workers. The KH-induced cation leak was (a) rapid in onset, (b) of a magnitude higher in the first ten minutes of treatment than subsequently, (c) of a multi-hit nature, (d) unaffected by a variety of membrane-active agents, and (e) inhibited by Zn2+, Cd2+ or mixing of toxin with dibutyl phthalate. Neuraminidase treatment of HRBC enhanced KH-induced cation influx and haemolysis, suggesting that additional receptors for TDH were unmasked by this treatment. In the presence of subhaemolytic KH, physiological levels of extracellular Ca2+ increased K+ influx by the Gardos channel, and Mg2+ (1.5 mM) decreased flux by the Na+/KV2Cr cotransporter. There were no significant changes to sodium pump activity. TDH and El Tor haemolysin (ETH) were purified from culture supernatants of V. parahaemolyticus and non-Ol V. cholerae, respectively. Highly purified TDH was derived from KH and used to confirm that the identified features of KH action were attributable to TDH. Although ETH also caused haemolysis by colloid osmosis secondary to increased cation permeability, it differed from TDH in its lability to air/vibration, relative magnitude of induced influx with respect to time, and selectivity series of induced lesion. Concentrated supernatants, from V. cholerae strains deleted of known virulence factors, caused morphological changes of Chinese hamster ovary cells, suggesting the presence of unidentified factors capable of perturbing cell physiology. Confirming its potential as an enterotoxin, KH (albeit at a high dose) increased the efflux rate constant for 86Rb+ from rabbit jejunocytes. These findings are discussed in the context of a possible pathogenic role for TDH in the gastrointestinal tract.

Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1983. / Typescript (photocopy).

Thesis (M.S.)--Catholic University of America. / Includes bibliographical references.

No description available.

No description available.

Thesis - Univ. of Michigan.

Thesis (Ph. D.)--New York University, 1929. / Bibliography: p. 787, 235.

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

Treponema hyodysenteriae is the causative agent of swine dysentery, a mucohemorrhagic disease of the intestines. T. hyodysenteriae requires phospholipids and cholesterol (or cholestanol) for growth, and it produces a a-hemolysin (TH) which is induced (300 fold) by the addition of RNA core to cultures. TH is bound to the RNA core which acts as a carrier or stabilizer. I\lyobjectives were to (i) obtain successful continuous growth of T. hyodysenteriae; (ii) study the production of the hemolysin produced by T. hyodysenteriae; (Ui) study the effects of different oligonucleotide carriers on the induction of hemolysin, (iv) examine various purification procedures for nipid, efficient partial purification of the hemolysin, (v) separate the hemolysin from the RNA core carrier, and (vi) to detennme whether T. hyodysenteriae can use coprostanol, the most common intestinal sterol, to satisfy its sterol requirement. I found that optimal growth of T. hyodysenteriae could be achieved by using BHI- glucose broth supplemented with calf serum (I O~/o). serum replacement (100/0), or phosphatidylcholine liposomes which contained cholesterol or cholestanol. Optimal growth required 1 % 02 and stirring of the culture. l\laximal hemolytic titers were obtained during late-log to early-stationary phase by cultures grown in the presence of RNA core. Hemolysin production was induced as soon as 5 minutes after addition of RNA core to cultures. This production was inhibited by chloramphenicol. Polyguanylic acid and RNase treated RNA core did not significantly increase hemolytic titers of cultures grown in their presence. Partial purification of hemolysin was achieved by acetic acid clarification followed by ammonium sulfate precipitation (65% saturation). With these procedures > 90% of the hemolytic activity was recovered. Although, hydroxylapatite adsorption and polyethyleneimine precipitation completely adsorbed or precipitated hemolytic activity I I was unable to efficiently recover the activity. Partially purified hemolysin was c}10toxic to CHO cells, and caused lysis rather than the rounding effect caused by many cytotoxins. / Master of Science

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1977. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Vita. / Includes bibliographical references. / Ph.D.