Listeria monocytogenes überwindet endotheliale Barrieren, um eine Meningitis oder Encephalitis auszulösen. Das Hindurchtreten durch diese Barriere könnte über die Invasion von Endothelzellen durch Listerien aus dem Blut und anschließender Freisetzung der Bakterien ins Gehirn erfolgen. In den ersten Infektionsmodellen, in denen gezeigt wurde, daß Listerien in der Lage sind Endothelzellen zu invadieren, wurden humane, makrovaskuläre Nabelschnurendothelzellen (HUVEC) verwendet. Die für die Ausbildung der Blut-Hirn-Schranke verantwortlichen mikrovaskulären Hirnendothelzellen (BMEC) unterscheiden sich aber deutlich von den makrovaskulären HUVEC. In der vorliegenden Arbeit wurde die Interaktion von L. monocytogenes mit HUVEC und mit humanen BMEC (HBMEC) untersucht. Es konnte gezeigt werden, daß L. monocytogenes HBMEC effizient invadieren kann. Nach der Aufnahme und dem Entkommen der Bakterien aus dem Phagosom bilden sie Aktinschweife aus, mit deren Hilfe sie sich im Zytoplasma frei bewegen können. Listerien sind in der Lage, sich in HBMEC über einen Zeitraum von 20 Stunden zu vermehren und über eine Ausbreitung von Zelle zu Zelle in benachbarte Zellen zu gelangen. Mit einem Listerien-Stamm, der das grün-fluoreszierende Protein (GFP) exprimiert, konnte der Infektionsverlauf in HBMEC über einen Zeitraum von 20 Stunden in Echtzeit verfolgt werden. Hierbei zeigte sich, daß auch stark infizierte HBMEC sich nicht vom Untergrund ablösen oder lysieren und somit gegenüber intrazellulären Listerien sehr widerstandsfähig sind. Wie rasterelektronenmikroskopische Aufnahmen von HBMEC-Monolayern nach einer Infektion mit L. monocytogenes erkennen ließen, adhärieren Listerien an HBMEC, indem sie einen engen Kontakt mit Mikrovilli auf HBMEC eingehen. Mit Listerien infizierte HBMEC bilden wenige Stunden nach der Infektion Membranausstülpungen aus, in denen sich Listerien befinden. Diese Ausstülpungen sind mit der Zelle nur noch über sehr dünne Membranschläuche verbunden. Um herauszufinden, welche Listerienproteine an der Aufnahme von L. monocytogenes in HUVEC und HBMEC beteiligt sind, wurden verschiedene Deletionsmutanten auf ihre Invasivität in HUVEC und HBMEC getestet. In Gegenwart von 20 Prozent Humanserum wurden HUVEC in einer von den Oberflächenproteinen InlA, InlB und ActA unabhängigen Weise von L. monocytogenes invadiert. Wurde das Gen, welches für den positiven Regulationsfaktor PrfA kodiert, deletiert, reduzierte dies die Invasionsrate beträchtlich. Listerienstämme mit einer Deletion im für InlB kodierenden Gen sind unfähig, HBMEC zu invadieren. Neben InlG und ActA spielt auch PrfA eine entscheidende Rolle bei der Invasion von L. monocytogenes in HBMEC. Die Adhäsion von L. monocytogenes an HBMEC ist von InlB unabhängig. Auch die apathogene und nicht-invasive Art L. innocua bindet an HBMEC. Humanserum hemmt die Invasion von L. monocytogenes in HBMEC, nicht aber in HUVEC. Während sich die Invasionsraten von L. monocytogenes in HUVEC durch Zentrifugation bei der Infektion erhöhen ließen, hatte die Zentrifugation keine Auswirkung auf die Invasivität von L. monocytogenes in HBMEC. Neben diesen konnten in dieser Arbeit noch weitere Infektionsparameter gefunden werden, die unterschiedliche Auswirkungen auf die Invasion von L. monocytogenes in HUVEC und HBMEC haben. Im Zellüberstand von HUVEC konnten bis zu 6 Stunden nach einer Infektion mit L. monocytogenes große Mengen an IL-8 nachgewiesen werden. Während eine Infektion von HUVEC mit L. monocytogenes die Expression von IL-6-spezifischer mRNA schwach induzierte, war keine vermehrte Expression von MCP-1- und VCAM-1-spezifischer mRNA feststellbar. Indem Caco-2-Zellen und HBMEC auf gegenüberliegenden Seiten eines Filters bis zur Konfluenz kultiviert wurden, konnte ein in-vitro-Modell des choroid plexus etabliert werden. Wenige Stunden nach der Infektion von HBMEC mit L. monocytogenes befanden sich auch in den Caco-2-Zellen Listerien. Wie elektronenmikroskopisch nachgewiesen werden konnte, waren diese Listerien durch die Filterporen in die Epithelzellen gelangt. Der Mechanismus, dem diese Ausbreitung zugrunde liegt, ist noch unbekannt. / Listeria monocytogenes cross endothelial barriers to cause meningitis or encephalitis. Passage through the barrier may be achieved by invasion of endothelial cells by Listeria from the blood stream followed by release of the bacteria into the brain. Internalization of L. monocytogenes by endothelial cells has been previously demonstrated using macrovascular human umbilical vein endothelial cells (HUVEC) as a model system. However, brain microvascular endothelial cells (BMEC) which form the blood brain barrier, are strikingly different from the macrovascular HUVEC. Therefore, in this investigation, the interaction of L. monocytogenes with HUVEC and human BMEC (HBMEC) was studied. It was found that L. monocytogenes efficiently invades HBMEC. After invasion and escape from the phagosome the bacteria induce the formation of actin tails which allows them to move intracellularly. Once within the HBMEC, L. monocytogenes are able to multiply over a period of at least 20 hours and enter neighbouring cells by cell-to-cell spread. Using a green fluorescent protein-expressing L. monocytogenes strain, this process of infection was followed in real time. It was shown that heavily infected HBMEC do not detach from the tissue culture dish and do not lyse, indicating that they are highly resistant to intracellular L. monocytogenes. Scanning electron microscopy studies of infected HBMEC-monolayers showed adherent Listeria in close contact with surface microvilli. Listeria-infected HBMEC shows bacteria-containing membrane protrusions within a few hours after infection. These protrusions are connected with the cell suface via thin stalk-shaped membrane connections. To determine which listerial proteins are responsible for the uptake of L. monocytogenes in HUVEC and HBMEC, different deletion mutants of L. monocytogenes were tested with respect to their effect on the efficiency of invasion. It was found that L. monocytogenes invades HUVEC in the presence of 20 per cent human serum independently of InlA, InlB, and ActA. However, deletion of the gene encoding the positive virulence regulatory factor PrfA results in a strong inhibition of invasion. Listeria-strains with a deletion in the InlB encoding gene are unable to invade HBMEC. Moreover, InlG, ActA, and also PrfA play important roles in invasion of HBMEC by L. monocytogenes. Adherence of L monocytogenes to HBMEC is independent of InlB. Even the nonpathogenic and noninvasive species L. innocua adheres to HBMEC. Human serum was shown to inhibit the uptake of L. monocytogenes by HBMEC but not by HUVEC. Centrifugation of Listeria onto the cells enhanced the invasion of HUVEC, but had no effect on invasion of HBMEC. In addition to these differences, other parameters were identified which have different effects on the invasiveness of L. monocytogenes for HUVEC and HBMEC. High amounts of IL-8 could be detected in the supernatants of Listeria-infected HUVEC within 6 hours. Additionally, a weak induction of IL-6 specific mRNA could be detected during infection of HUVEC, but mRNA-expression specific for MCP-1 and VCAM-1 was not altered. Using HBMEC and Caco-2 cells, an in-vitro-model of the choroid plexus was developed by growing each cell type on either side of porous membrane filters. A few hours after infection of HBMEC with L. monocytogenes, bacteria were found in the underlying Caco-2 cells. In transmission electron microscopic studies it could be shown that Listeria reached the epithelial cells via the filter pores. The mechanism for this spreading is so far unknown.

Listeriae are Gram positive, facultative, saprophytic bacteria capable of causing opportunistic infections in humans and animals. This thesis presents three separate lines of inquiries that can lead to the eventual convergence of a global view of Listeria as pathogen in the light of evolution, genomics, and function. First, we undertook to resolve the phylogeny of the genus Listeria with the goal of ascertaining insights into the evolution of pathogenic capability of its members. The phylogeny of Listeriae had not yet been clearly resolved due to a scarcity of phylogenetically informative characters within the 16S and 23S rRNA molecules. The genus Listeria contains six species: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi; of these, L. monocytogenes and L. ivanovii are pathogenic. Pathogenicity is enabled by a 10-15Kb virulence gene cluster found in L. seeligeri, L. monocytogenes and L. ivanovii. The genetic contents of the virulence gene cluster loci, as well as some virulence-associated internalin loci were compared among the six species. Phylogenetic analysis based on a data set of nucleic acid sequences from prs, ldh, vclA, vclB, iap, 16S and 23S rRNA genes identified L. grayi as the ancestral branch of the genus. This is consistent with previous 16S and 23S rRNA findings. The remainder 5 species formed two groupings. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch within this group. Deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri support the proposed tree. This implies that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri; and that pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. Second, we attempted to reconstitute L. innocua of its deleted virulence gene cluster, in its original chromosomal location, from the L. monocytogenes 12 Kb virulence gene cluster. This turned out particularly difficult because of the limits of genetic tools presently available for the organism. The reconstitution was partially successful. The methods and approaches are presented, and all the components necessary to complete the constructs are at hand for both L. innocua and the parallel, positive control of L. monocytogenes mutant deleted of its virulence gene cluster. Third, the sequencing of the entire genome of L. monocytogenes EGDe was undertaken as part of an EU Consortium. Our lab was responsible for 10 per cent of the labor intensive gap-closure and annotation efforts, which I helped coordinate. General information and comparisons with sister species L. innocua and a close Gram positive relative Bacillus subtilis are presented in context. The areas I personally investigated, namely, sigma factors and stationary phase functions, are also presented. L. monocytogenes and L. innocua both possess surprisingly few sigma factors: SigA, SigB, SigH, SigL, and an extra-cytoplasmic function type sigma factor (SigECF). The stationary phase genes of L. monocytogenes is compared to the well-studied, complex, stationary phase networks of B. subtilis. This showed that while genetic competence functions may be operative in unknown circumstances, non-sporulating Listeria opted for very different approaches of regulation from B. subtilis. There is virtually no overlap of known, stationary phase genes between Listeria and Gram negative model organism E. coli. / Listerien sind Gram-positive, fakultativ intrazelluläre, saprophytische Bakterien, die in der Lage sind, bei Mensch und Tier opportunistische Infektionen hervorzurufen. Die vorliegende Arbeit veranschaulicht drei unterschiedliche Versuchsansätze, die schließlich hinsichtlich Evolution, Genom- und Funktionsanalysen zur Konvergenz der globalen Sichtweise von Listeria als pathogener Mikroorganismus führen können. Zunächst wurden phylogenetische Analysen durchgeführt, die einen Einblick in die Evolution des pathogenen Potentials von Mitgliedern der Gattung Listeria geben sollten. Aufgrund mangelnder phylogenetischer Informationen bezüglich der 16S und 23S rRNAs war eine genaue phylogenetische Entschlüsselung der Gattung Listeria jedoch nicht möglich. Die Gattung Listeria umfaßt sechs verschiedene Spezies: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri und L. grayi, wobei L. monocytogenes und L. ivanovii zu den pathogenen Bakterien zählen. Die Pathogenität wird hierbei durch ein 10-15 kb großes Virulenzgencluster determiniert, das in L. monocytogenes, L. ivanovii sowie in L. seeligeri vorzufinden ist und neben einigen Virulenz-assoziierten Internalin-Genen zum genetischen Vergleich der sechs verschiedenen Spezies herangezogen wurde. Die im Rahmen der phylogenetischen Analysen untersuchten Nukleinsäuresequenzen der Gene prs, ldh, vclA, vclB, iap sowie die der 16S und 23S rRNA-Gene deuteten darauf hin, daß L. grayi dem gemeinsamen Vorläufer der Gattung Listeria am nächsten steht, was mit den bisher verfügbaren 16S und 23S rRNA-Daten übereinstimmt. Die verbleibenden fünf Spezies bilden zwei Gruppen, die sich zum einen aus L. monocytogenes und L. innocua und zum anderen aus L. welshimeri, L. ivanovii und L. seeligeri zusammensetzen. Die Positionen der im Virulenzgencluster von L. innocua und L. welshimeri identifizierten Deletionen bestätigen den hier vorgeschlagenen Stammbaum, was darauf hindeutet, daß im gemeinsamen Vorfahren von L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri und L. welshimeri das Virulenzgencluster vorhanden war und daß das pathogene Potential in L. innocua bzw. in L. welshimeri durch zwei unabhängige Ereignisse verloren ging. Weiterhin wurde versucht, das 12 kb-Virulenzgencluster von L. monocytogenes in der ursprünglichen Lokalisation in das Chromosom der Spezies L. innocua, die eine Deletion des Virulenzgenclusters aufweist, zu integrieren. Dieser Ansatz erwies sich aufgrund der derzeit limitierten Methodik zur genetischen Manipultation dieses Organismus als sehr problematisch und führte bisher nur teilweise zum Erfolg. Die angewandten Strategien zur Klonierung der erforderlichen Konstrukte, die zur Erstellung der beschriebenen L. innocua-Mutante und einer L. monocytogenes-Mutante mit Deletion des Virulenzgenclusters erforderlich sind, werden vorgestellt. Der dritte Schwerpunkt der Arbeit befaßte sich mit der vollständigen Sequenzierung des Genoms von L. monocytogenes EGDe, die im Rahmen eines EU-Konsortiums durchgeführt wurde. Unser Labor war zu 10 Prozent an der Lückenschließung zwischen den Sequenz-Contigs sowie an der Annotierung beteiligt, für deren Koordinierung ich verantwortlich war. Vergleiche der Genomsequenzen von L. monocytogenes, L. innocua und dem verwandten Gram-positiven Bakterium Bacillus subtilis werden vorgestellt, wobei ein besonderer Schwerpunkt auf den Genen für Sigma-Faktoren und Stationärphase-Funktionen liegt. Sowohl L. monocytogenes und L. innocua enthalten mit SigA, SigB, SigH und einem extrazytoplasmatischen Sigma-Faktor, SigECF, überraschend wenige Sigma-Faktoren. Die Stationärphase-Gene von L. monocytogenes werden mit dem gut untersuchten, sehr komplexen Stationärphase-System von B. subtilis verglichen. Dies zeigte, daß, obwohl genetische Kompetenz unter nicht bekannten Umständen eine Rolle spielen könnten, in Listeria völlig unterschiedliche Mechanismen der Genregulation wirksam sind. Es ist keinerlei Überlappung mit den bekannten Stationärphase-Genen des Gram-negativen Modellorganismus Escherichia coli festzustellen.

Listeria monocytogenes, ein fakultativ intrazellulärer Krankheitserreger, besitzt die Fähigkeit, Wirtszellen zu penetrieren, sich in ihnen zu vermehren, sich intrazellulär zu bewegen und auch benachbarte Zellen direkt zu infizieren. Die intrazelluläre Fortbewegung erfolgt durch Polymerisation von zellulärem Aktin, wodurch charakteristische Aktinschweife an einem Pol der Bakterien entstehen. Der einzige bakterielle Faktor, der für die Aktinpolymerisation notwendig ist, ist das Oberflächenprotein ActA. ActA allein ist aber nicht in der Lage, Aktin zu polymerisieren, sondern kann dies nur in Assoziation mit Proteinen der Wirtszelle. Die einzigen bisher bekannten Wirtszellproteine, die direkt mit ActA interagieren, sind das Phosphoprotein VASP und der Arp2/3-Komplex. VASP bindet an den zentralen prolinreichen Bereich von ActA und beschleunigt durch die Rekrutierung von Profilin den Prozeß der Aktinpolymerisation. Der Arp2/3-Komplex interagiert mit dem N-terminalen Bereich von ActA und initiiert die eigentliche Aktin-Polymerisation. Um weitere eukaryotische, mit ActA interagierende Proteine (AIPs) zu isolieren, wurde über einen "Yeast Two-Hybrid"-Test mit ActA als Köder eine embryonale Maus-cDNA-Genbank getestet. Dabei wurden drei verschiedene AIPs identifiziert, von denen eines identisch mit dem humanen Protein LaXp180 (auch "CC1" genannt) ist. LaXp180 ist ein 180 kDa Protein mit über 50 theoretischen Phosphorylierungsstellen in der N-terminalen Hälfte, während die C-terminale Hälfte "coiled-coil"-Strukturen ausbilden kann. Darüberhinaus enthält LaXp180 eine Kern-Lokalisations-Sequenz und ein Leucin-Zipper-Motiv. Die Bindung von LaXp180 an ActA wurde in vitro unter Verwendung von rekombinantem His6-Tag-LaXp180 und rekombinantem ActA bestätigt, da rekombinantes ActA nur an einer Ni-Agarose-Säule gebunden wurde, wenn diese vorher mit His6-Tag-LaXp180 beladen war. Über RT-PCR konnte zum ersten Mal die Expression LaXp180-spezifischer mRNA in verschiedenen Säugerzellen nachgewiesen und mit einem polyklonalen anti-LaXp180-Serum durch Immunopräzipitation erstmals ein 194 kDa großes Protein in Säugerzellextrakten detektiert werden. Die intrazelluläre Lokalisation von LaXp180 wurde über Immunfluoreszenzmikroskopie untersucht. Immunfluoreszenzfärbungen von Fibroblasten mit dem anti-LaXp180-Serum zeigten eine starke Färbung der Zellkerne und definierter Bereiche direkt neben den Kernen, während das restliche Zytoplasma schwach gefärbt war. Über Immunfluoreszenzmikroskopie mit dem anti-LaXp180-Serum an mit L. monocytogenes infizierten Zellen konnte gezeigt werden, daß LaXp180 mit der Oberfläche vieler, aber nicht aller intrazellulärer, ActA-exprimierender Listerien kolokalisiert. Dagegen wurde nie eine Kolokalisation mit intrazellulären, aber ActA-defizienten Mutanten beobachtet. Darüberhinaus ist LaXp180 asymmetrisch auf der Bakterienoberfläche verteilt und schließt sich gegenseitig mit der F-Aktin-Polymerisation aus. LaXp180 ist ein putativer Bindungspartner von Stathmin, einem 19 kDa Phosphoprotein, das die Mikrotubuli-Dynamik reguliert. Über Immunfluoreszenz konnte gezeigt werden, daß auch Stathmin mit intrazellulären, ActA-exprimierenden L. monocytogenes kolokalisiert. / Listeria monocytogenes, a facultative intracellular pathogen, is able to penetrate and to multiply in host cells, to move intracellularly and to infect neighbouring cells directly. Intracellular movement is caused by the polymerisation of host cell actin leading to the generation of characteristic actin tails at one pole of the bacterium. The only bacterial factor necessary for actin polymerisation is the surface protein ActA. However, ActA itself is unable to polymerize actin, and can only do so in association with host cell proteins. So far the only known host cell proteins directly interacting with ActA are the phosphoprotein VASP and the Arp2/3 complex. VASP binds to the central proline-rich repeat region of ActA and accelerates actin polymerisation by the recruitment of profilin. The Arp2/3 complex interacts with the N-terminal domain of ActA and initiates the actin polymerisation process. To identify additional eukaryotic ActA-interacting proteins (AIPs), an embryonic mouse cDNA library was screened in a yeast two-hybrid approach using ActA as bait. Three different AIPs were isolated, one of which was identical to the human protein LaXp180 (also called "CC1"). LaXp180 is a 180 kDa protein with more than 50 theoretical phosphorylation sites in the N-terminal part. The C-terminal part is able to form coiled-coil structures. Furthermore, LaXp180 contains a nuclear localisation sequence and a leucine-zipper motif. Binding of LaXp180 to ActA was demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Recombinant ActA was retained on a Ni-agarose column after prior loading of the column with histidine-tagged LaXp180. Using RT-PCR, the expression of LaXp180 specific mRNA in different mammalian cells was demonstrated for the first time. A protein with a molecular weight of 194 kDa was detected in cell extracts by immunoprecipitation with a polyclonal anti-LaXp180 antiserum. The intracellular localisation of LaXp180 was analysed by immunofluorescence microscopy. Immunofluorescence staining of fibroblasts with the anti-LaXp180 serum showed a strong staining of the nuclei and of defined regions next to the nuclei as well as a weak staining of the rest of the cytoplasm. Fluorescence microscopy with the anti-LaXp180 antiserum of cells infected with L. monocytogenes revealed colocalisation of LaXp180 with the bacterial surface of a subset of intracellular, ActA-expressing listeriae. In contrast, colocalisation with intracellularly growing but ActA-deficient mutants was never observed. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerisation on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a 19 kDa phosphoprotein regulating microtubule dynamics. Using immunofluorescence, colocalisation of stathmin with intracellular, ActA-expressing L. monocytogenes was also demonstrated.

The primary focus of this dissertation is the metabolic enzyme pyruvate carboxylase (PC). The structure and function of this fascinating enzyme has been studies and characterized by many laboratories over many decades. This extensive background is reviewed in Chapter 1, with an overview of the biotin-dependent carboxylase family and a particular focus on PC. In this dissertation, we primarily use X-ray crystallography to study PC at a structural level. This dissertation is divided into two overarching sections, with the first section (Chapters 2-5) focusing on the bacterial second messenger cyclic-di-AMP (c-di-AMP). This project was initiated by our collaborators in the laboratory of Josh Woodward at the University of Washington, who performed the first screen to identify c-di-AMP binding proteins in the bacterium Listeria monocytogenes. In Chapters 2 and 3, the regulation of PC by c-di-AMP in L. monocytogenes as well as the bacterium Lactococcus lactis is discussed. Crystal structures of the PC from each of these species in complex with cyclic-di-AMP reveal the binding site and give insights into the molecular mechanisms of this regulation. In Chapters 4 and 5, structural studies of other c-di-AMP binding proteins identified in the screen are discussed. The second section (Chapters 6) focuses on a second class of PC enzymes called the two-subunit PCs, which are found in a subset of Gram-negative bacteria. In Chapter 6, the first crystal structure of a two-subunit PC from the bacterium Methylobacillus flagellatus is determined. In collaboration with the Lars Dietrich laboratory at Columbia University, we investigate the physiological function of the two-subunit PC in the pathogen Pseudomonas aeruginosa. A theme which emerges from these studies is that PC is an incredibly diverse enzyme which has been adapted for the peculiar physiological needs of each organism it inhabits. Because PC is found throughout nature in every kingdom of life, further studies of its unique properties and role in each organism are sure to provide more surprising insights in the years to come.

Señala que la contaminación microbiana de alimentos durante la cadena de producción es una de las principales causas de enfermedades transmitidas por alimentos (ETAS). Listeria monocytogenes, es un microorganismo patógeno causante de la listeriosis humana, una enfermedad con alta morbilidad, hospitalización y mortalidad, principalmente en poblaciones vulnerables. Su presencia en alimentos es favorecida por su resistencia a bajas temperaturas y amplio rango de pH; los brotes están vinculados a carnes frescas y procesadas, productos lácteos y alimentos listos para comer. En los últimos años los bacteriófagos han sido propuestos para su uso con fines terapéuticos y como una alternativa para el control biológico de Listeria monocytogenes y otros microorganismos patógenos en alimentos. Los bacteriófagos son virus que infectan a las bacterias, se componen de una cadena simple o una cadena doble de ADN o ARN cubiertos por una cápside proteica y están presentes casi en todos los ecosistemas. A partir de un filtrado de aguas servidas de camales de la ciudad de Lima, se seleccionó, aisló y caracterizó fisicoquímica y biológicamente un bacteriófago lítico para Listeria monocytogenes: el fago ASCF1. Este bacteriófago es estable a temperaturas entre 4°C y 45°C, es tolerante a un amplio rango de pH (desde 4 hasta 9), presenta un amplio rango de hospedero, infectando también cepas de Salmonella typhimurium y Vibrio cholerae. Tiene una multiplicidad de infección (MOI) óptima de 10, un periodo de latencia de 120 minutos y un tamaño de explosión promedio de 9.72 unidades formadoras de placa por célula infectada. Estos resultados indican que el bacteriófago ASCF1 puede ser aplicado para la descontaminación de alimentos y superficies. / Tesis

There is a constant need of new synthetic emulsifiers in the food industry. Sugar esters are widely used as food grade synthetic emulsifiers, amongst which sucrose esters are the most common. Although sucrose esters are used very frequently, little is known about the use of lactose esters in food. There is a need for characterization of lactose esters before they can be used in foods. The objective of this study was to characterize a lactose ester, lactose monolaurate (LML) as an antimicrobial agent on food pathogens, evaluate its effect on 20 % oil-in-water emulsions as an emulsifier, and to explore its effect on crystallization behavior of anhydrous milk fat. In the first study (Chapter 3), the effect of LML was evaluated on survival of some Gram-positive and Gram-negative bacteria. For Listeria monocytogenes, a concentration of 1 mg/ml showed some inhibition in growth media whereas the cells were completely killed at 5 mg/ml. For Mycobacteria, an LML concentration between 0.1-1mg/ml was lethal. Scanning electron microscopy was also conducted to examine any changes in the morphology of cells. Listeria exhibited a change in morphology and a wrinkling effect was shown in Mycobacteria. In the second study (Chapter 4), the effect of LML as an emulsifier was evaluated in 20 % oil-in-water emulsions. The use level of LML was comparable to commercially available emulsifier polysorbate 20, and produced comparable stabilization in the emulsions upon use. In this study, an attempt was also made to optimize the synthesis of LML with respect to the immobilized enzyme and solvent combination. It was concluded that for 20 % oil-in-water emulsions, LML is a promising emulsifier at 0.5%. In the third study (Chapter 5), the effect of LML was evaluated at two concentrations on the crystallization behavior of anhydrous milk fat at two temperatures with high and low supercooling. On application of high intensity ultrasound (HIU) to anhydrous milk fat (AMF) at 31°C and 0.05 % LML the effect on viscosity of sample and crystallization behavior was evaluated. It was concluded that the viscosity of AMF decreased with the addition of 0.05% LML. The lower viscosity of anhydrous milk fat on addition of LML could be restored with the application of HIU.

L. monocytogenes is ubiquitous in environment and can grow and survive in a wide range of environmental conditions. It contaminates foods via raw materials or food processing environments. However, the current knowledge of its ecology and in particular, the mode of environmental survival and transmission of L. monocytogenes remains limited. One important aspect of environmental survival of L. monocytogenes may be contact with other microorganisms, including amoebae, which naturally feed on bacteria as a source of nutrients. In this context, research has shown that several intra-cellular pathogens are able to survive or replicate within free-living amoebae. In view of the potential for amoebae to act as environmental reservoirs for bacteria, the interaction of L. monocytogenes with freeliving Acanthamoeba spp. and the impact of plasmid-associated genes on their interaction were investigated. Several strains of environmental and clinical isolates of L. monocytogenes were used for co-culture with amoebae. Axenic amoebae were isolated from environmental sources (water, soil) by cultivation in PYG supplemented with antibiotics. L. monocytogenes strains were co-cultured with amoebae on plates, in trays (as monolayers of amoeba cells) and in flasks to provide qualitative and quantitative assessments of the survival of bacteria and amoebae. Bacteriological methods and microscopy (fluorescence, TEM and phase contrast) were used to track the fate of internalized bacteria. A vector that allowed GFP expression under control of the prfA promoter was used to assess the expression of Listeria virulence genes within amoeba cells. The role plasmid encoded genes in interactions of L. monocytogenes with amoebae was assessed using plasmid-cured versus wild type in the co-cultures. Finally the mechanisms of bacterial uptake and killing by A. polyphaga were assessed using chemical inhibitors that affected actin polymerization (Cytochalasin D, Wortmannin), phagosome-lysosome fusion (Suramin) and phagosomal acidification (ammonium chloride, Bafilomycin A and Monensin). Sequence analysis of a section of the large plasmid from strain DRDC8 revealed a high level of similarity of gene organization and DNA sequences with plasmid-borne genes found in other Listeria spp. and Gram-positive bacteria. While the majority of environmental isolates of L. monocytogenes contained a large plasmid, it was absent in clinical isolates. Further, plasmid of DRDC8 was lost during serial passage in HeLa cells. This data indicated that the plasmid may be readily lost during isolation procedures or during growth within host animals/cells and thus plasmid instability may explain the absence of plasmid in clinical isolates. Co-culture of L. monocytogenes with Acanthamoeba spp. showed these amoebae are able to actively phagocytose and kill bacteria within 2-5 h irrespective of temperature used. Amoebae killed both plasmid cured and LLO mutants with the same rate for the parental bacteria. Fluorescence microscopy and TEM of bacteria within trophozoites showed the bacteria become confined within tight vacuolar structures surrounded by lysosomes and mitochondria and degraded after 4 to 5 h post phagocytosis. This data indicated that although L. monocytogenes is an effective pathogen of mammalian cells, it could not escape from phagosomes and evade the killing mechanisms of amoeba trophozoites. Consequently, bacteria are killed within phagolysosome in trophozoites before they express their virulence genes to escape from phagosomes and get access to cytoplasm. This was confirmed by observing no GFP expression by bacteria carried prfA::gfp construct in co-culture with amoebae whereas it was observed during co-culture with HeLa cells. Using inhibitors, the mechanisms involved in phagocytosis, and killing of L. monocytogenes cells by A. polyphaga were assessed. The results showed that the uptake of bacterial cells is mediated by the trophozoites but not the bacteria and mannose binding protein is not involved in this process. Furthermore, pre-treatment of trophozoites with inhibitors indicated both phagosomal acidification and phagosome lysosome fusion are involved in killing of bacteria. These results suggest that compared to mammalian cells e.g. HeLa cells, amoeba trophozoites are better able to effectively inactivate and destroy internalized L. monocytogenes cells. In conclusion, Acanthamoeba spp. are able to uptake and kill L. monocytogenes cells in phagolysosome compartments. However, bacteria can saprophyticaly grow on materials released from amoeba trophozoites. Thus this group of amoebae is not able to harbour L. monocytogenes cells, or act as environmental reservoirs for this opportunistic pathogen under the laboratory conditions tested. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292868 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth. Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites. To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins. L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes. Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

The bacterium Listeria monocytogenes can cause the disease listeriosis in both humans and animals. For the epidemiological investigation of listeriosis detection and characterisation of the organism are important steps. Paper I. There are few reports on the incidence of L. monocytogenes in clinical samples from humans in India. Therefore, we investigated 144 samples from immunocompromised patients. L. monocytogenes was isolated from two placental bits from women with poor obstetric history, one patient with renal failure and three other patients. Five isolates were positive for the virulence genes hlyA, actA and iap. The sixth isolate was positive for hlyA and actA genes. Paper II. Characterisation of 601 human L. monocytogenes isolates causing invasive listeriosis during the period 1986 to 2007 in Sweden reveals a decrease in serovar 4b strains. Since 1996, serovar 1/2a has become the predominant serovar causing human listeriosis: PFGE analysis revealed two clusters including different serotypes suggesting that we need more studies on genetic relatedness among clinical isolates. Paper III. The incidence of Listeria species in seafood from markets in Goa was studied. One hundred and fifteen raw/fresh seafoods bought at the fish markets were sampled and tested for presence of Listeria spp. L. monocytogenes was detected in 10 samples. L. monocytogenes in raw seafood may pose a health risk in kitchen if contaminating ready-to-eat food. Paper IV. Gravad and cold-smoked salmon are associated with human listeriosis in Sweden. L. monocytogenes was isolated from 11 of 56 products. Serovar 1/2a was predominant, followed by 4b. REA/PFGE typing of the isolates identified five types of L. monocytogenes. One type was identical to a human type, two other were closely related.These findings suggest that gravad and cold-smoked salmon are still possible sources of listeriosis in Sweden. Paper V. Many outbreaks of listeriosis have been related to consumption of dairy-associated products. Therefore, 123 farm bulk milk samples in India and 20 cervico-vaginal samples from dairy cows with reproductive disorders were investigated for L. monocytogenes. L. monocytogenes was isolated from 17.9% of bulk milk samples and from 10% of cervico-vaginal swabs. The virulence gene hlyA was detected in all isolates. These findings represent a public health risk where unpasteurised milk and milk products are largely consumed. Paper VI. Isolates of L. monocytogenes (n=177) from 22 animal species were characterized and compared with human strains isolated between 1986-2006 in Sweden. Although many animals and humans shared pulsovars, they did not appear at the same time or with the same proportion of strains. The pulsovars shared by both animals and humans may indicate that there is an exchange of L. monocytogenes strains between these two groups due to either direct or indirect transmission. / <p>The work is done in cooperation with the School of Hospitality, Culinary Arts &amp; Meal Science, Örebro UniversityVishal Singh Parihar, Örebro University, Department of Restaurant and Culinary Arts, Sörälgsvägen 2, SE-712 60 Grythyttan, Sweden or ICAR Research Complex for Goa, Ela, Old Goa – 403402, Goa, India. Phone 0832-2284678/79; Fa:0832-2285649. E-mail: drvishu@yahoo.co.in</p>

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found. My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.