In vitro assays were developed using small intestinal contents from broilers fed maltodextrin in preparation for shipping. This was done to establish an effective bactericidal dose of allicin or lysozyme as ingredients in maltodextrin-based feed. The antimicrobials were added to overnight cultures of gut material bacteria from maltodextrin fed broilers and a pure culture of Salmonella. Following this, lysozyme was incorporated into maltodextrin feed at 0, 10 and 20 g.kg-1 of feed and offered for 9 h to 4 pens of 20 birds per treatment. Bacterial numbers were analyzed using Proc Mixed of SAS. Allicin and lysozyme inhibited Enterobacteriaceae and Clostridium perfringens, respectively, in vitro. Lysozyme showed the most promise; it reduced bacterial numbers in nutrient broth. Feeding lysozyme-enriched maltodextrin for 9 h inhibited bacilli growth (P<0.05) when evaluated using next generation sequencing. Lysozyme was effective in reducing specific bacterial numbers in the gut of market-aged broilers / The project focused on ways to ensure poultry meat safety by controlling bacteria population in the gastrointestinal tract of market-aged broilers prior to shipping.

Introduction: Garlic-derived organosulfur compounds are associated with physiological benefits, including the reduction of cardiovascular disease (CVD) risk, possibly by reducing the risk marker trimethylamine-N-oxide (TMAO). TMAO production in humans is largely influenced by the metabolic activity of the intestinal bacteria on dietary precursors including L-carnitine. Dietary supplementation of bioactive garlic phytochemical allicin has recently been suggested to reduce the formation of TMAO precursor molecule trimethylamine (TMA) from L-carnitine through impact on the intestinal bacteria, thereby limiting the formation of TMAO by the host. Purpose: The objective of this research was to evaluate and compare the efficacy of fresh and aged garlic extracts (rich in alliin and allicin, respectively) in the reduction of circulating TMAO levels produced from L-carnitine metabolism and identify shifts in the abundances of gastrointestinal bacterial genes that may contribute to reduction in circulating TMA levels, which may, in turn, influence the levels of circulating TMAO. Methods: Five-week old female C57BL/6 mice (n = 12) were challenged with L-carnitine to assess the animal's capacity for TMAO production. Animals were gavaged daily with fresh or aged garlic extract dissolved in L-carnitine for 13 days, then challenged with L-carnitine post-treatment to evaluate changes in TMAO production. Whole blood samples were evaluated for TMAO content using UPLC-MS/MS and compared to non-extract consuming control groups. Post-mortem hepatic tissues were collected and analyzed for TMA-oxidizing flavin monooxygenase 3 (Fmo3) gene abundance and protein expression using quantitative real-time PCR (qPCR) and ELISA. Fecal samples collected prior to and following treatment were analyzed using qPCR to quantify shifts in the abundance of L-carnitine metabolizing genes cntAB and grdH. Results: Postprandial and circulating TMAO levels were not significantly affected (p < 0.05) by inclusion of garlic extract in the diet. Dietary intervention with extracts significantly increased L-carnitine-derived proatherogenic CVD risk marker γ-butyrobetaine levels ~28% higher than the increased levels observed in the positive control group supplemented with L-carnitine only. Mice administered garlic extracts had significant increases of, γ-butyrobetaine, relative to negative control mice and mice supplemented with broad-spectrum antibiotics. Mice supplemented fresh garlic extract saw a 25-fold increase in circulating γ-butyrobetaine levels after intervention; mice supplemented aged garlic extract saw a 23-fold increase in circulating γ-butyrobetaine levels after intervention. Furthermore, FMO3 protein expression levels in either extract treatment group were not significantly different (p < 0.05) from controls. Abundances of L-carnitine metabolizing genes in fecal samples of mice fed either garlic extract were not significantly higher than levels observed in positive or negative controls. Interestingly, treatment with broad-spectrum antibiotics significantly increased abundances of L-carnitine metabolizing genes cntAB and grdH when compared with controls. Abundances of hepatic Fmo3 mRNA transcript in mice supplemented garlic extracts were not significantly different from the positive control group when data were normalized to mg of liver used. Mice supplemented aged garlic extracts significantly lowered Fmo3 mRNA transcript levels relative to the negative control. Significance: This research suggests that garlic extract supplementation in conjunction with excess L-carnitine consumption may not be an appropriate dietary intervention strategy to reduce CVD risk. As it stands, garlic extract supplementation may increase CVD risk by promoting the biosynthesis of proatherogenic γ-butyrobetaine. The impact of garlic extract mediated increases in γ-butyrobetaine should be further investigated in tandem with CVD outcomes to confirm the findings presented in this study. / Doctor of Philosophy / Garlic compounds that contain sulfur are associated with many health benefits, including the reduction of heart disease risk, possibly by lowering the amount of risk marker trimethylamine-N-oxide (TMAO) in the body. TMAO is produced when the gut bacteria break down L-carnitine into trimethylamine (TMA), which is then absorbed and converted to TMAO in the liver. Garlic supplementation has recently been suggested to reduce TMAO formation, which may, in turn, reduce heart disease risk. The objective of this research was to evaluate the potential of fresh and aged garlic extracts (which have different sulfur compounds in them) to reduce TMAO levels and identify changes in the gut bacteria that may contribute to this lowering effect. Mice were fed daily with either fresh or aged garlic extract for 13 days, then given L-carnitine to evaluate changes in TMAO levels in the blood. These levels were then compared to mice that did not consume any garlic extract. Liver samples were tested for their ability to turn TMA into TMAO. Fecal samples were tested to determine if there were any changes to gut bacteria caused by the garlic extracts. TMAO levels in the mice were not significantly affected by consuming garlic extracts. Consuming garlic extracts did, however, increase another risk marker of heart disease known as γ-butyrobetaine. Feeding mice garlic extracts did not affect the ability of mice to turn TMA into TMAO, nor did it affect the gut bacteria. This research suggests that garlic extracts may not be an appropriate strategy to reduce heart disease risk. As it stands, garlic extract supplementation may increase heart disease risk by promoting the γ-butyrobetaine formation. The means that garlic extracts increase γ-butyrobetaine levels should be further investigated.

The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite the current challenges in NP research, these molecules are still relevant today as they are a major source of human medicine as well as being useful biological tools. The elucidation of their biosynthetic pathways has also provided information about the biochemical and biophysical properties of fascinating enzyme families such as the α- oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’- phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several important NP, including those essential for life. This study reports the characterization and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E. coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is responsible for its characteristic yellow colour. It binds L-serine as a natural substrate with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray crystallography at a resolution of 4.98 Å, which shows four molecules in the asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal region where the ACP domain is located in the crystal strucuture also suggests intrinsic flexibility and disorder within that region. With the increasing demand for new anti-infective therapies, investigations of the molecular interactions between NPs and their protein targets are vital in understanding the inhibition or activation properties of these molecules. The cysteine transpeptidases known as sortases produced by Gram positive bacteria have been identified as attractive targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray crystallography. This study reports the first high resolution crystal structure of the H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain. Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow, tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6 μM. The interaction resulted in a covalent adduct with the active site cysteine residue (Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to 250 μM with an efficacy cut-off point at higher concentations. These results indicate that chalcone flavonoids are worth further investigation as potential antibiofilm inhibitors. A renewed interest in plant NPs has also led to a collaborative investigation on the antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh preparations of garlic extracts. This investigation reports the first evidence that allicin and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE) against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin (BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit further investigation as adjuncts to existing antibiotics.

Alliin is a predominant flavor precursor in garlic cloves. It interacts with the enzyme alliinase when garlic cloves are crushed, cut, or chewed to produce allicin, an unstable thiosulfinate that is the main biologically active component of fresh crushed garlic. Biological functions and health benefits of garlic include reduction of cancer risk in humans, improving immune system, and anti-microbial, anti-oxidant, and anti-hypertensive activities. The quality of fresh garlic and garlic products is usually related to its alliin content and allicin release potential. This research presents a simple, rapid, and precise HPLC method for alliin determination. It involves the use of 30:70% methanol: water and 0.05% sodium dodecylsulfate mobile phase composition, C18 5 μm disc column of size 3.9 x 150 μm, and detector set at 210 nm. The method showed good reproducibility with 0.56%-4.11% relative standard deviations, a linear response of peak area to alliin concentration of 0.4 ng/mL-80 ng/mL, and average recovery of 93.5%-101%. Determination of alliin in eight garlic samples indicated the highest amount in garlic tablet that was expected. The method presented is economical and efficient and can be used in alliin determination. The method gave a satisfactory chromatograms with methanol-hydrochloric acid extract but not with hot water extract.

Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

Zusammenfassung Einleitung: Eimeria tenella ist ein obligat intrazellulärer Parasit und Erreger der Blinddarmkokzidiose beim Huhn. Aufgrund des verbreiteten Einsatzes von Antikokzidia sind seit langem Resistenzentwicklungen bekannt. Die zugrundeliegenden Mechanismen sind komplex und nicht genau bekannt. Für den Nachweis von Resistenz gilt der Tierversuch als „Goldstandard“, er ist aber aufwändig, zeitraubend und ethisch problematisch. Ziele der Arbeit: Es sollte ein für die Erstellung von Sensitivitätsprofilen und Wirksamkeitsprüfungen geeignetes In-vitro-Modell für E. tenella entwickelt und validiert werden. Weiterhin sollte auf mit Antikokzidiaresistenz assoziierte Änderungen im Proteom von Kokzidien untersucht werden. Material, und Methoden: Monolayer von MDBK (Madin-Darby bovine kidney cells) dienten zur Kultivierung von E. tenella für den in vitro ASA (anticoccidial sensitivity assay). Als Parameter wurden die Inhibition (%ISIA, %IRIA) der Invasion durch Sporozoiten (SIA = sporozoite invasion inhibition assay) und der Erregerreproduktion (RIA = reproduction inhibition assay) mittels quantitativer PCR (qPCR) für E. tenella beurteilt. Die minimale Hemmkonzentration (MIC = minimal inhibitory concentration) wurde anhand einer Referenz (sensitiver Laborstamm Houghton, Ref-1) für Monensin (Mon), Salinomycin (Sal), Maduramicin (Mad), Lasalocid (Las) und Toltrazuril (Tol) bestimmt. Auf dieser Basis wurden ein zweiter Laborstamm (Wisconsin, Ref-2) und vier Feldstämme (T-376, FS-1, FS-2 und FS-3) im SIA und RIA getestet. 1.0 × 105 (SIA) und 5.0 × 104 (RIA) Sporozoiten/Well wurden verwendet. Die in vitro Ergebnisse wurden mit den in vivo Daten verglichen. Alle in vitro Versuche wurden in vier Replikaten durchgeführt. Insgesamt wurden 420 Hühner (1. Lebenstag: LT, Cobb-500) in fünf Tierversuche verwendet, um die Empfindlichkeit der E. tenella-Stämme gegen verschiedene Antikokzidia zu untersuchen. In jedem Tierversuch wurden 84 Tiere (12. LT) in 7 Gruppe (n = 12) eingeteilt. Hühner wurden mit 7,5 × 104 Oozysten je Tier am 14. LT infiziert. Die Wirksamkeitsprüfung für den alternativen Naturstoff Allicin erfolgte mittels RIA am Stamm Ref-1. Kontinuierlich in vitro als Tachyzoiten kultivierte T. gondii des RH-Stammes (Sen-RH) wurden unter ansteigenden Mon-Dosen in HFF-Zellen (human foreskin fibroblasts) über 8 Monate auf Resistenz (MonR-RH) selektiert. Es erfolgte eine stabile Isotopenmarkierung (SILAC). Für E. tenella wurden die sensitivenReferenzstämme ebenso wie die Feldstämme variabler Sensitivität isoabarisch-chemisch markiert (Tandem-Massenmarkierung, TMT). Die quantitativen Proteomanalysen erfolgten mit dem nanoUPLC-MS/MS. Ergebnisse: Für Ref-1 betrugen die MIC95 (MIC für eine 95%ige Inhibition) 0,25 (Mon), 0,5 (Sal), 1,0 (Mad), 0,5 (Las) und 5,0 (Tol) μg/ml. Die in vitro Sensitivitätsprofile für Ref-2, FS-1, FS-2, und FS-3 stimmten für die Ionophoren mit den Ergebnissen des Tierversuchs gut überein, für Tol war das aber nicht der Fall. Signifikante Korrelationen wurden für die %IRIA-Werte mit dem Oozystenindex (OI, r = 0,290-0,507; p < 0,05), dem lesion score (LS, r = 0,279-0,345; p < 0.05, für Ref-1, Ref-2 und FS-1) und der Gewichtszunahme im Tierversuch (WGNNC, r = 0.284-0.419; p < 0.05, für Ref-1 und FS-1) festgestellt. Allicin in einer Konzentration von 1,8 mg/ml ergab einen %IRIA von 99,0%, was einer guten Wirkung entspricht. Mittels SILAC wurden 1820 Proteine identifiziert, von denen 1024 Proteine in mehr als vier biologischen Replikaten quantifizierbar waren. Bei 52 Proteinen wurden signifikante Unterschiede zwischen Sen-RH und MonR-RH festgestellt (p < 0,05). Actin, beta-Tubulin cofactor D, Perforin-like Protein 1 (PLP1), und kleine GTPase-vermittelte Signaltransduktionsproteine (GTPases) wurden in T. gondii MonR-RH hochreguliert (p < 0,05). Insgesamt waren 42 Proteine bei der resistenten Mutante MonR-RH hochreguliert. Für E. tenella wurden 97 Proteine identifiziert und 25 Proteine wurden in allen drei biologischen Replikaten quantifiziert. Mon-resistente E. tenella zeigten signifikant hochreguliertes Actin (p < 0,05). Mikronemenproteine wurden in resistenten Toxoplasma (MIC8) und Eimeria (MIC4) herunter reguliert. Schlussfolgerungen: Das entwickelte In-vitro-Verfahren in Kombination mit der qPCR eignet sich zur Bewertung der Sensitivität von E. tenella gegenüber ionophoren Antikokzidia und erbringt Ergebnisse, die mit dem Tierversuch korrelieren. Das Modell kann Tierversuche zum Screening von Wirkstoffen reduzieren, aber nicht vollständig ersetzen, vor allem, wenn die Wirkung (auch) späte Entwicklungsstadien im Zyklus von E. tenella betrifft. Letzteres könnte die mangelnde Aussagekraft für Tol erklären. In T. gondii lässt sich Resistenz in vitro induzieren. Die Proteom-Analyse zeigte eine Verminderung der Invasion (↑ Actin, ↓ MIC8) und des Austritts (↓ PLP1), und eine Aktivierung der Replikation (↑ Actin, ↑ beta-Tubulin cofactor D, und ↑ GTPases proteine). Mon führt zu einer Erhöhung des intrazellulären Na+ gefolgt von einer Erhöhung der Ca++ Konzentrationen. Die Reduzierung der Expression von Proteinen in MonR-RH, die mit Invasion- und Austrittsaktivitäten zusammenhängen, zeigt an, dass dieser Stamm höhere Konzentrationen von Mon verträgt, indem er die ktitische Schwelle der zytosolischen Ca++ Konzentration erhöht ist, die erforderlich ist, um diese Prozesse zu provozieren.