Most studies on innate immunity in ticks have focused on the antimicrobial peptides from hemolymph, such as defensins and lysozyme, while less is known about bacterial recognition molecules, or antimicrobial mechanisms in other tissues. The current study attempted to identify novel antimicrobial mechanisms, with a focus on bacterial recognition by hemolymph proteins and antimicrobial activity in salivary gland extracts. Using bacteria as affinity beads, two high molecular mass molecules (Protein X and Protein Y) have been identified in tick hemolymph. These proteins are thought to interact with the bacterial surface via ionic interactions. Tandem mass spectrometry analysis followed by de novo sequencing indicated that these proteins are novel as no homologs could be identified from sequence databases. In an attempt to clone Protein X, using a degenerate primer obtained from a de novo sequence, an unrelated hemocyte protein was identified. This protein, named savicalin, was shown to belong to the lipocalin family based on bioinformatical analysis. Transcriptional profiling indicated that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. To date, this is the first tick lipocalin not derived from salivary glands. Interestingly, up-regulation of its mRNA transcript in response to bacterial challenge suggests that this protein could be involved in antimicrobial activity. Up-regulation after feeding also suggests a role in the post-feeding development of the tick. Two different approaches were used to purify the Gram-positive antibacterial activity from salivary gland extracts. The first attempt entailed a two-step separation approach. Tricine SDS-PAGE of the active fraction showed 3 components (~20, ~10 and ~7 kDa). BLAST searches using the N-terminal sequences of the latter proteins identified the ~20 kDa protein as savignin, while the other two proteins could not be matched. The second strategy included an ultrafiltration step (10 kDa cut-off) and MS-analysis of the active fraction in this case indicated the presence of various components with molecular masses ranging from 0.99 – 7.182 kDa, with 12 predominant components ranging from 0.99 - 4.448 kDa. Further tandem mass spectrometry analysis of the active fraction revealed the presence of three tick actin-derived fragments. This is of interest as actin fragments have been implicated in innate immunity of other invertebrates. In this study, synthetic peptides corresponding to one of the detected tick actin fragments as well as actin5C (detected in Drosophila hemolymph) were found not to inhibit the growth of Bacillus subtilis when tested up to a concentration of 100 ìg/ml. It is envisaged that future studies of immunoprotective molecules of the tick, O. savignyi, may contribute to the development of novel anti-infective agents and potential targets for anti-tick vaccine design. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

La qualité microbiologique d’un produit cosmétique se doit d’être conservée durant la totalité de sa durée de vie grâce à l’ajout notamment de conservateurs antimicrobiens. Néanmoins, les conservateurs organiques traditionnellement utilisés sont particulièrement décriés depuis quelques années car suspectés d’effets secondaires. Dans l’objectif de disposer d’alternatives à ces substances, les propriétés antimicrobiennes de l’oxyde de zinc (ZnO) ont été étudiées. L’efficacité antimicrobienne de ces particules inorganiques a ainsi été évaluée sur les cinq micro-organismes d’intérêt pour l’industrie cosmétique (E. coli, S. aureus, P. aeruginosa, C. albicans et A. brasiliensis). Pour cela des tests microbiologiques ont été mis en place en milieu gélosé et bouillon liquide pour évaluer la sensibilité de chaque souche microbienne au ZnO. L’efficacité de ces poudres a été évaluée au sein de diverses formules cosmétiques via la réalisation de Challenge Tests. Des études spécifiques visant à améliorer la compréhension des mécanismes antimicrobiens du ZnO ont été menées : les phénomènes de dissolution des particules générant des cations zinc, de production photochimique de radicaux libres ou encore de contact direct entre particules et cellules microbiennes ont été approfondis. Couplés à des études supplémentaires visant à affiner les relations structure/activité, ces travaux ont été menés dans l’objectif d’optimiser le potentiel antimicrobien de ces poudres pour la présente application. Ce projet a démontré que le ZnO permettait de conserver la qualité microbiologique de produits dermopharmaceutiques variés, allant des émulsions aux poudres. Dépendamment de sa concentration, les actions bactéricides, levuricide et fongistatique du ZnO confèrent aux produits la capacité de répondre aux exigences requises en termes de conservation. Les poudres minérales de ZnO apparaissent alors comme une alternative appropriée aux conservateurs organiques / The microbiological quality of a cosmetic product should be preserved during its whole shelf-life notably thanks to the addition of antimicrobial preservatives. Nevertheless, commonly used organic preservatives are particularly criticized since a few years because they are suspected of side effects. In order to propose alternatives to these substances, the antimicrobial properties of zinc oxide (ZnO) were studied. The antimicrobial efficacy of these inorganic particles was evaluated on the five microorganisms of interest for the cosmetic industry (E. coli, S. aureus, P. aeruginosa, C. albicans and A. brasiliensis). Microbiological tests were designed in agar medium and liquid broth to evaluate the sensitivity of each microbial strain to ZnO. The efficacy of these powders was evaluated in various dermopharmaceutical formulations via Challenge Tests. Some specific studies dedicated to improve the understanding of antimicrobial mechanisms of ZnO were carried out: (i) particles dissolution generating zinc cations, (ii) photochemical generation of free radicals (iii) direct contact between particles and microbial cells. Coupled with additional studies designed to refine structure/activity relationships, this work was performed in order to optimize the antimicrobial potential of these powders for the present application. All these studies demonstrated that ZnO enabled the preservation of the microbiological quality of various cosmetic products (emulsions and powders). The bactericidal, levuricidal and fongistatic activities of ZnO were dependent of its concentration and confer to the products the ability to comply with the demands in term of preservation. The inorganic powders of ZnO appear as suitable alternatives to organic preservatives

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.