The aim of the study is to investigate the causal relationship between membrane-damaging activity and antibacterial action of cardiotoxins from Naja naja atra (Taiwan cobra) cardiotoxin 3 (CTX3) and Naja nigricollis (Egyptian cobra) toxin gamma. Compared with that on Escherichia coli (E. coli, Gram-negative bacteria), CTX3 showed a greater growth inhibition on Staphylococcus aureus (S. aureus, Gram-positive bacteria). Antibacterial avtivity of toxin gamma toward E. coli and S. aureus was similar. Bactericidal action of cardiotoxins positively correlated with increase in membrane permeability of bacterial cells. Morphological examination showed that cardiotoxins disrupted the integrity of bacterial membrane. Cardiotoxins showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall promoted bactericidal effect of cardiotoxins on E. coli and S. aureus, respectively. CTX3 notably permeabilized model membrane of S. aureus and toxin gamma had similar activity on the permeabilization of bacterial model membrane used. Membrane-damaging activity of cardiotoxins was inhibited by either LPS or LTA, while increasing concentrations of cardiotoxins counteracted the inhibitory action of LPS and LTA. Oxidation of Met residues on loop II of cardiotoxins simultaneously attenuated membrane-permeabilizing activity and bactericidal effect of cardiotoxins. Taken together, our data indicate that antibacterial action of cardiotoxins depend on their ability to induce membrane permeability.

B rucella lipopolysaccharide is an important component of virulence in brucellosis. Recent research in macrophage models has shown that Brucella LPS does not behave like classical LPS by stimulating potent inflammatory responses. The central hypothesis of this work is that O-antigen is dynamic signaling molecular and participates in complex interactions with the host to promote productive infection. A corollary to this is that the host environment is dynamic, and Brucella has evolved mechanisms to cope with changing environments. In an effort to understand the contribution of Brucella LPS to virulence and pathogenesis, the function of a metabolic locus important in the synthesis of LPS has been demonstrated and complemented. The spontaneous loss of LPS expression has been characterized. Contribution of LPS to acquisition of the host environment in tissue culture and mouse models has been explored. This work demonstrated that genes outside the O-antigen biosynthesis ( manBA) cluster contribute to LPS biosynthesis. Further high frequency mutation involving manBA is partly responsible for observed dissociation of Brucella strains. Finally, work herein attempts to look at the role of LPS in acquisition of the host environment and shows that LPS is important for recruiting particular cell populations within a host model of brucellosis.

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The autonomic nervous system serves to maintain a constant inner environment, a process termed homeostasis. Thus, in response to the homeostatic challenge posed by infectious agents, the autonomic nervous system answers to signals from the immune system and elicits adaptive physiological and behavioral reactions. These so called sickness responses include fever, anorexia, hyperalgesia, social avoidance, and the release of stress hormones. Neuropeptides, used in the communication between neurons, are because of their release properties and sustained actions likely mediators of homeostatic responses. The enkephalinergic system constitutes one of the largest neuropeptidergic systems in the brain, but its involvement in inflammatory conditions has been little studied. We first examined the immune-induced activation of the parabrachial nucleus (paper I), an enkephalinergic autonomic relay center in the brain stem. We found that intravenous injection of bacterial endotoxin, lipopolysaccharide (LPS), activated the external lateral parabrachial subnucleus, as measured in terms of Fos expression, but that the enkephalinergic cell population in this subnucleus was largely separated from the LPS-activated neurons. Because Fos may not always be a reliable activity marker, we next examined by in situ hybridization the immune-induced expression of newly transcribed preproenkephalin (ppENK) heteronuclear RNA (hnRNA), which gives a direct indication of the utilization of enkephalin in a particular neuron (paper II). We detected induced expression of ppENK hnRNA in several autonomic structures in the brain, including the paraventricular hypothalamic nucleus (PVH) but not the parabrachial nucleus, indicating increased enkephalinergic signaling activity in the positively labeled structures during inflammatory condition. We then examined the projections of the immune-induced ppENK transcribing PVH neurons by injecting rats intraperitoneally with the retrograde tracer substance Fluoro-Gold, hence labeling neurons with axonal projections outside the blood-brain barrier, followed by systemic injection of LPS (paper III). Dual-labeling histochemical and hybridization techniques showed that the vast majority of the ppENK hnRNA expressing cells were hypophysiotropic cells, hence being involved in neuroendocrine regulation. These findings suggest that centrally produced enkephalin is involved in the coordination of the sickness responses during systemic immune challenge, including the modulation of the release of stress hormones or other hypothalamic hormones during inflammatory conditions. We next turned to the role of prostaglandins in the hypothalamic-pituitary-adrenal (HPA) axis response to inflammation. We injected mice deficient for the terminal prostaglandin (PG) E2 synthesizing enzyme mPGES-1 with LPS and studied their stress hormone release (paper IV). The genetically modified mice displayed attenuated plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone during the later phases of the HPA-axis response compared with wild type mice, and this impairment did not depend on a changed activation pattern in the brain, but instead correlated to an early decrease in corticotropin-releasing hormone mRNA expression in the PVH, hence being the likely cause of the blunted ACTH and corticosterone responses at later time-points. Based on these findings we suggest that a neural, mPGES-1-independent pathway, and a humoral, mPGES-1-dependent pathway act in concert but in distinct temporal patterns to initiate and maintain the HPA-axis response during immune challenge. In addition to activating the central limb of the HPA-axis, inflammatory mediators have been suggested to act directly on the adrenal gland to induce the release of corticosterone, but little is known about the underlying mechanisms. We examined adrenal tissue isolated from rats injected with LPS or interleukin-1β (IL-1β) (paper V), and found that immune stimulation resulted in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes were accompanied by an induced production of IL-1β and IL-1 receptor type 1, as well as of cyclo-oxygenase-2 and mPGES-1 in these cells, implying local cytokine-mediated PGE2 production in the adrenals, which also displayed EP1 and EP3 receptors in the cortex and medulla. Additional mechanistic studies using an IL-1 receptor antagonist showed that IL-1β acts locally to affect its own synthesis, as well as that of cyclooxygenase-2. Taken together these data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.

The hallmark of Gram-negative bacteria is the presence of an outer membrane (OM) surrounding the cytoplasmic membrane (here called the inner membrane [IM]) and the cell wall. The OM is a unique asymmetric bilayer with an inner leaflet consisting of phospholipid and an outer leaflet consisting of lipopolysaccharide (LPS). LPS is a large anionic molecule that typically contains six fatty acyl chains and up to several hundred sugar residues. This chemical structure explains why the OM is relatively impermeable to large hydrophobic molecules, such as detergents, bile salts, and high molecular weight antibiotics, which readily cross a normal phospholipid bilayer. LPS and the OM are essential to the viability of most Gram-negative organisms, including major human pathogens. LPS molecules are biosynthesized at the IM and subsequently exported out of the IM, across the intermembrane space (the periplasm) and through the OM to their final position at the cell surface. In Escherichia coli, the essential LPS transport proteins, LptA-G, are required for this process. This Lpt pathway includes an IM adenosine triphosphate binding cassette (ABC) transporter, LptBFG, which is associated with an additional IM protein, LptC; a periplasmic protein, LptA; and an OM complex consisting of the lipoprotein LptE and the transmembrane \(\beta\)-barrel protein LptD. All seven Lpt proteins associate as a single complex that spans the cell envelope. However, little is known about how these proteins work together to transport LPS. Here, we use in vivo and in vitro biochemical studies to probe the organization, function, and assembly of the Lpt machine. In Chapter 2, we show that LptE forms a plug within the LptD \(\beta\)-barrel and present a model for how this unusual structure can move LPS from the periplasm directly into the outer leaflet of the OM. In Chapter 3, we demonstrate that the Lpt transenvelope bridge consists of a series of structurally homologous domains – LptC, LptA, and the N-terminal domain of LptD – stacked in a head-to-tail orientation, providing a route for LPS from the IM to the OM. Finally, in Chapter 4, we connect these two sets of results by showing how the assembly of the Lpt transenvelope bridge is regulated by that of the LptD/E complex in the OM. Together, these findings explain how the functions of the Lpt proteins are coordinated to ensure delivery of LPS to the correct cellular compartment. A fundamental understanding of LPS biogenesis will contribute to the development of new therapies against Gram-negative infections.

No description available.

We investigated the effects of dietary folic acid and age, on immunological parameters in laying hens challenged with LPS. 48 Shaver White hens at different ages were fed 2 wheat-soybean based diets with 0 or 4 mg supplemental FA per kg of diet for 8 wk. 6 hens from each dietary treatment were injected with 8 mg/kg body weight of LPS or saline and observed for 4 h. A few diet X challenge interaction were observed in young hens, whereas FA and LPS independently influenced a number of parameters. In older hens, there were very few diet X challenge interactions as well as effects of FA, whereas LPS affected several parameters. In conclusion, dietary FA influenced some immune responses in younger hens, but no such effects were observed in older hens. FA may modulate immune responses in laying hens under acute LPS challenge which could be tissue and age dependent.

We investigated the effects of dietary folic acid and age, on immunological parameters in laying hens challenged with LPS. 48 Shaver White hens at different ages were fed 2 wheat-soybean based diets with 0 or 4 mg supplemental FA per kg of diet for 8 wk. 6 hens from each dietary treatment were injected with 8 mg/kg body weight of LPS or saline and observed for 4 h. A few diet X challenge interaction were observed in young hens, whereas FA and LPS independently influenced a number of parameters. In older hens, there were very few diet X challenge interactions as well as effects of FA, whereas LPS affected several parameters. In conclusion, dietary FA influenced some immune responses in younger hens, but no such effects were observed in older hens. FA may modulate immune responses in laying hens under acute LPS challenge which could be tissue and age dependent.

Lipopolysaccharide (LPS), one of the main components of Gram-negative bacterial cell walls, is a potent endotoxin. Structures of the unique protective monoclonal antibody (mAb) WN1 222-5 in complex with Escherichia coli R2 and R4 LPS core regions show that recognition occurs in a manner similar to the innate immune receptor Toll-like receptor 4 (TLR4). Inner core LPS is shown to exist in a conserved epitope with multiple intramolecular interactions that allows the conserved epitope to bind strongly to mAb WN1 222-5. The structure of mAb FDP4, directed against truncated E. coli J-5 LPS, shows a deep pocket combining site specific for a terminal epitope that does not allow room for wild type (wt) LPS. Research into these anti-LPS binding mAbs opens up new avenues for potential septic shock therapy. The explosion of new techniques and bright x-ray sources in the 80’s and 90’s led to rapid advancement of protein x-ray crystallography; however, structure determination on some of the most important problems is now stalled due to the general inability to grow crystals of sufficient size. Recent advances in electron microscopy (EM) technology has led to improved beam characteristics, which has allowed the initiation of research to develop EM as a viable alternative to x-ray crystallography. In this research, method development using standard equipment to explore potential avenues for analysing three-dimensional protein crystals via EM has been explored. / Graduate / 0982 / 0487 / 0537 / kgomery@uvic.ca

The sympathetic nervous system (SNS) is part of an integrative network that functions to restore homeostasis following injury and infection. The SNS provides negative feedback control over inflammation through the secretion of catecholamines from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Central autonomic structures receive information regarding the inflammatory status of the body and reflexively modulate SNS activity. Evidence suggests that inflammation and infection can also directly regulate ACC function. However, the precise alterations in ACC function that occur in response to regional inflammation, systemic inflammation and exposure to bacterial products have yet to be fully characterized. The present thesis was therefore performed to test the hypothesis that gastrointestinal (GI) and systemic inflammation modulate ACC Ca2+ signaling, and that ACCs possess the ability to directly detect microbe-associated molecular patterns (MAMPs). Ca2+ signaling was assessed in single ACCs isolated from control mice and mice with GI or systemic inflammation using Ca2+ imaging and perforated patch clamp electrophysiology. Acute and chronic GI inflammation consistently reduced high-K+-stimulated Ca2+ transients in ACCs through an inhibition of voltage-gated Ca2+ current. In contrast, systemic inflammation significantly enhanced high-K+-stimulated Ca2+ transients and catecholamine secretion through an increase in Ca2+ release from the endoplasmic reticulum. Incubation of control ACCs in serum obtained from mice with systemic inflammation produced a similar increase in Ca2+ signaling, suggesting that circulating mediators play an important role in this response. To determine whether ACCs can directly detect MAMPs, Ca2+ signaling, excitability and neurotransmitter release were assessed in control ACCs and ACCs incubated in media containing lipopolysaccharide (LPS). Unlike GI and systemic inflammation, LPS did not affect ACC Ca2+ signaling. However, LPS dose- and time-dependently hyperpolarized ACC resting membrane potential and enhanced large conductance Ca2+-activated K+ currents. Consistent with membrane hyperpolarization, LPS reduced ACC excitability and inhibited neuropeptide Y release. These effects were mediated by Toll-like receptor 4 and nuclear factor-κB. In summary, GI and systemic inflammation produce opposite effects on ACC Ca2+ signaling through distinct mechanisms, and ACCs can directly detect MAMPs. These findings extend our knowledge of the complex integration performed by the immune system-nervous system network during health and disease. / Thesis (Ph.D, Physiology) -- Queen's University, 2013-08-20 17:15:23.945