Nos últimos anos, vegetais têm sido responsáveis por surtos de enfermidades transmitidas por alimentos (ETA) em diversas regiões do mundo por serem veículos dos mais diferentes micro-organismos patogênicos, entre eles Salmonella spp, Listeria monocytogenes e Escherichia coli produtora de toxina de Shiga (STEC). O uso de sanitizantes nem sempre reduz de maneira significativa a população de micro-organismos presentes nos vegetais, sendo necessária a aplicação de técnicas mais eficientes, entre elas, a radiação gama. Assim, os objetivos do presente estudo foram avaliar o efeito da irradiação na redução de STEC, Salmonella spp. e L. monocytogenes inoculadas em espinafre minimamente processado, bem como sobre os atributos físico-químicos e sensoriais do vegetal. Amostras de espinafre (Tetragonia expansa) foram inoculadas com um \"pool\" de cepas de Salmonella spp, um \"pool\" de cepas de L. monocytogenes e um \"pool\" de cepas de STEC, separadamente, e expostas às doses de 0; 0,2; 0,4; 0,6; 0,8 e 1,0 kGy. Os valores de D10 para Salmonella spp, L. monocytogenes e STEC foram, respectivamente, 0,19 a 0,20 kGy, 0,20 a 0,21 kGy e 0,17 kGy. Foram avaliados os comportamentos de Salmonella spp, L. monocytogenes e STEC em amostras de espinafre expostas à doses cinco vezes maiores do que o valor D10 obtido para cada micro-organismo: 1,0; 1,05 e 0,85 kGy, respectivamente, e em amostras não irradiadas armazenadas por 12 dias a (4±1) °C e a (10±1) °C. Os resultados mostram que as doses empregadas reduziram a população de Salmonella e de STEC em aproximadamente 6 ciclos log no dia zero tendo permanecido abaixo do limite de detecção (<10 UFC/g), mesmo após 12 dias de armazenamento em ambas as temperaturas. A dose de 1,05 kGy reduziu a população de L. monocytogenes em, aproximadamente, 5 log imediatamente após a irradiação, porém com recuperação de 2,62 log nas amostras armazenadas a (10±1) °C ao final do período de armazenamento. Amostras de espinafre expostas às doses de 1 e 1,5 kGy e a amostra-controle, mantidas sob refrigeração (4±1) ºC, foram utilizadas para a avaliação da vida de prateleira (VP), análise sensorial, análise de cor, determinação de ácido ascórbico, flavonoides, compostos fenólicos e capacidade antioxidante. A VP da amostra exposta à dose de 1 kGy foi de 15 dias, dois dias a mais que a da amostra-controle, enquanto a exposta a 1,5 kGy apresentou VP de 12 dias. Todas as amostras expostas à radiação foram aceitas pelos provadores. A irradiação não provocou alterações significativas na concentração de compostos fenólicos e atividade antioxidante, porém houve alteração na cor e na concentração de flavonoides. As estações do ano, por sua vez, tiveram influência sobre a coloração, concentração de compostos fenólicos e atividade antioxidante. Apesar da alteração na coloração ter sido observada na análise instrumental, esta não foi percebida pelos provadores durante a análise sensorial. O processo de irradiação mostrou ser uma boa alternativa para aumentar a segurança microbiológica de espinafre sem alterar as características sensoriais. No entanto, o uso das Boas Práticas de Fabricação nunca deve ser negligenciado. / In recent years, fresh produce have been responsible for foodborne disease outbreaks worldwide, due to their contamination by different pathogenic microorganisms such as Salmonella spp., Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC). The use of sanitizers does not always significantly reduce the microbial populations present in vegetables, and thus, the application of more efficient techniques such as gamma radiation, is required. The objectives of this study were to evaluate the effect of irradiation on the reduction of the populations of STEC, Salmonella spp. and L. monocytogenes, inoculated on minimally processed spinach, as well as to assess its effect on the sensory and physicochemical characteristics of the vegetable. Spinach (Tetragonia expansa) samples were individually inoculated, with a cocktail of three strains of Salmonella spp, three strains of L. monocytogenes and three strains of STEC and exposed to doses of 0, 0.2; 0.4; 0.6; 0.8 and 1.0 kGy. The D10 values determined in this study ranged from 0.19 to 0.20 kGy for Salmonella spp, 0.20 to 0.21 kGy for L. monocytogenes, and 0.17 kGy for STEC. The behavior of Salmonella spp., L. monocytogenes and STEC were evaluated in spinach samples exposed to doses of 1.0, 1.05 and 0.85 kGy, respectively, and in non-irradiated samples, stored for 12 days at (4±1) °C and (10±1) °C. The results showed that the populations of Salmonella and STEC were reduced at about 6 log, on day zero, and remained below the detection limit (<10 CFU/g) even after 12 days of storage at both temperatures tested. The 1.05 kGy dose reduced the population of L. monocytogenes in approximately 5 log, but in the samples stored at (10±1) °C, the growth of the microorganism (2,62 log) was observed at the end of the storage time. Spinach samples exposed to 1 and 1.5 kGy, as well as the control sample, all kept under refrigeration (4±1) °C were used for the evaluation of the product shelf life, sensory analysis, color analysis, determination of ascorbic acid, flavonoids, phenolic compounds and the antioxidant capacity. The samples exposed to 1 kGy displayed a shelf life of 15 days, two days longer than that observed for the control sample, while those exposed to 1.5 kGy showed a shelf life of 12 days. All samples exposed to radiation were accepted by the sensorial panel. The irradiation had no significant effect either on the concentration of phenolic compounds or on the antioxidant activity. Nevertheless, there was a reduction in the concentration of flavonoids and change on the color. The color, phenolic compounds concentration and antioxidant activity were influenced by the seasons of the year. Although the change in color was observed by instrumental analysis, this was not perceived by the panelists during sensory analysis. The irradiation process is a great alternative for microbiological safety purpose together with Good Manufacturing Practices.

O consumo mundial de produtos frescos tem aumentado nos últimos anos, devido a preocupação da população com a saúde. Entretanto, a busca por uma alimentação mais saudável vem aumentando o risco de surtos de doenças veiculadas por frutas e hortaliças, tendo em vista, que estes alimentos são geralmente consumidos in natura. Embora qualquer patógeno possa ser um problema potencial nesses produtos, Salmonella spp. tem grande relevância em saúde pública. A contaminação potencial por esse patógeno em frutas, é possível devido a contaminação cruzada e falta de boas práticas agrícolas durante o cultivo, colheita, manuseio e distribuição. Nas frutas de baixa acidez, dentre as quais está o caqui, Salmonella spp pode ainda ter condições para sobreviver e se multiplicar. O caqui é uma fruta de relevante importância sócio-econômica, com crescente demanda para exportação, no entanto, o mercado importador está cada vez mais exigente em relação a segurança alimentar. No Brasil, não existem informações sobre a ocorrência de Salmonella spp em caqui, bem como, a respeito do comportamento desse patógeno na polpa e na casca dessa fruta. Desse modo, os objetivos do presente estudo foram: (1) verificar a incidência de Salmonella spp na superfície do caqui, (2) estudar a sobrevivência e multiplicação de S. Enteritidis na polpa e na casca dessa fruta armazenada a diferentes temperaturas de incubação. Para tanto, em 2005 foram coletadas 77 amostras de caqui Fuyu e 80 amostras de caqui Rama Forte no varejo na cidade de Campinas-SP e a mesma proporção no ano de 2006. Adicionalmente em 2006 também foram coletadas 134 amostras de caqui Rama Forte e 134 amostras de caqui Fuyu no CEAGESP, na cidade de São Paulo. A pesquisa para Salmonella spp foi realizada pelo sistema BAX®, que se baseia na reação da polimerase em cadeia (PCR). Os parâmetros de crescimento de S. Enteritidis (duração da fase lag, taxa de crescimento exponencial e tempo de geração) foram obtidos dos dados experimentais. Os resultados mostraram que das 582 amostras analisadas 5 (0,9%) foram positivas para Salmonella spp, indicando que condições sanitárias durante o crescimento no campo (água de irrigação, adubo, etc.), colheita, condições de transporte e armazenamento, assim como a manipulação das frutas podem levar a contaminação. Os dados de crescimento obtidos para Salmonella mostram que este patógeno pode sobreviver e se multiplicar tanto na polpa quanto na casca do caqui, nas duas variedades estudadas e mesmo a baixas temperaturas. / The worldwide consumption of fresh produce has increased in the last years, due to an increased concern among the population about health. However, the search for more healthful eating habits has also increasing the risk of acquiring a foodborne disease, as these products generally are consumed raw. Although any pathogen can be a potential problem in these produces, Salmonella spp. is of great relevance in public health. The potential contamination for this pathogen in fruits, is possible due to cross contamination and poor agricultural practices during harvest, handling and distribution. In low acid fruits, amongst which is persimmon, Salmonella spp can still have conditions to survive and to multiply. The persimmon is a fruit of great economic importance, with increasing demand for exportation, however, the import market has an increase demand for food safety. There are not in Brazil, information about the occurrence of Salmonella spp in persimmon, as well as, information regarding the behavior of this pathogen in the pulp and the rind of this fruit. Thus, the objectives of the present study had been: (1) to verify the incidence of Salmonella spp on the surface of persimmon, (2) to study the survival and multiplication of S. Enteritidis on the peel and in the pulp of this fruit stored at different temperatures of incubation. For this survey in 2005 and 2006 harvests, 77 samples and 80 samples of \'Fuyu\' and \'Rama Forte\' persimmon respectively collected in the retail in Campinas city - SP. Additionally in 2006, 134 samples of each \'Rama Forte\' and \'Fuyu\' persimmon were also collected in the CEAGESP, in São Paulo city. The analyses had been carried using the BAX® system (Dupont/Qualicon), that is based on the Polymerase Chain Reaction (PCR). The parameters of growth during the evaluation of the ability of growth of S. Enteritidis (lag phase duration, exponential growth rate and generation time) were obtained from experimental data. Salmonella spp were detected in five samples (0,9%) of those analysed. The growth data obtained for Salmonella indicated that this pathogen can survive and multiply on the peel as well as in the pulp in the two studied varieties and even at low temperatures.

La salmonelosis de origen alimentario es una de las infecciones más importantes en humanos; a pesar de los controles en la cadena alimentaria, se sabe que su incidencia es cada vez mayor. Esto, sumado al riesgo de la resistencia antibiótica frente a Salmonella enterica genera una gran preocupación en salud pública. El objetivo del presente estudio fue detectar la presencia de Salmonella enterica en linfonódulos mesentéricos de cuyes y determinar el perfil de susceptibilidad antibiótica de las cepas obtenidas. Para ello se colectó el linfonódulo mesentérico craneal de 299 cuyes, provenientes de un matadero de la ciudad de Jauja, Junín. Estas muestras fueron procesadas y sembradas en caldos de enriquecimiento y medios de cultivo selectivos, de donde se obtuvieron las cepas. La identificación de las cepas de Salmonella enterica se realizó mediante pruebas bioquímicas. Posteriormente se evaluó la susceptibilidad de las cepas aisladas, por el método de Kirby Bauer, hacia antibióticos de uso común en medicina humana y veterinaria. De los 299 linfonódulos mesentéricos craneales evaluados, en 6 de ellos se aisló Salmonella enterica lo que representa un 2,0% ±1,5(6/299) de positividad. El 100% de estas cepas fueron sensibles a sulfatrimetropin, neomicina, gentamicina, ceftriazona y norfloxacina; el 84% a enrofloxacino y ácido nalidixico y 0% a furazolidona. Los resultados indican que la susceptibilidad a las quinolonas está disminuyendo, lo que puede ser un grave problema para la salud pública, además que el uso de furazolidona en casos de salmonelosis en cuyes es ineficiente. / Tesis

Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

6 Zusammenfassung In-vitro- und In-vivo-Studien zur Wirksamkeit von Eigelbantikörpern gegen Salmonella Enteritidis Michael Gürtler Institut für Lebensmittelhygiene der Veterinärmedizinischen Fakultät der Universität Leipzig Infektionen mit Salmonellen gehören weltweit zu den wichtigsten von Tieren auf den Menschen übertragbaren Erkrankungen. Anteilmäßig besitzen dabei die durch kontaminierte Lebensmittel, insbesondere Eier und Eiprodukte, hervorgerufenen Infektionen die größte Bedeutung. Dabei ist nach wie vor Salmonella Enteritidis (S. Enteritidis) in den Geflügelbeständen vorherrschend. Als Möglichkeit zur Bekämpfung von Salmonella-Infektionen bietet sich die passive Immunisierung der Legehennen durch orale Applikation von Eigelbantikörpern an. Bereits mehrfach wurde das Verfahren der Verabreichung von antikörperhaltigem Volleipulver zur Bekämpfung von bakteriellen Infektionskrankheiten, vor allem bei Kälbern und Ferkeln, getestet. Untersuchungen über die Einsatz solcher Eigelbantikörper mit dem Ziel der Zurückdrängung von Salmonellen in Legehennenbeständen sind bisher nicht bekannt geworden. Ziel dieser Arbeit war es, im Rahmen von In-vitro- und In-vivo-Studien den Einfluss von Eigelbantikörpern auf Vermehrung und Vorkommen von S. Enteritidis bei Legehennen zu untersuchen. Im Versuchskomplex A wurde in vitro ermittelt, ob Anti-S. Enteritidis-Antikörper im Eidotter in der Lage sind, die Vermehrung von Salmonellen zu hemmen. Im Versuchskomplex B wurde untersucht, ob es gelingt, durch die orale Verabreichung von Anti-S. Enteritidis-Eigelbantikörpern die Kontaminationsrate der gelegten Eier mit Salmonellen bei experimentell infizierten Legehennen zu beeinflussen. Im Versuchskomplex C sollte geprüft werden, ob Eigelbantikörper die Fähigkeit besitzen, das Adhäsions- und Invasionsvermögen von Salmonellen an bzw. in Epithelzellen einer permanenten Zelllinie zu beeinflussen. Im Rahmen von Versuchskomplex A wurden Legehennen mit einem inaktivierten S. Enteritidis-Stamm parenteral hyperimmunisiert, um hohe Konzentrationen an Antikörpern im Dotter zu induzieren. Die Eidotter wurden mit zwei S. Enteritidis-Stämmen in drei verschiedenen Kontaminationsdosen beimpft und das Vermehrungsverhalten durch Keimzählung als Generationszeit ermittelt. Im Versuchskomplex B wurden in zwei Infektionsversuchen Anti-S. Enteritidis-Eigelbantikörper in Form von Volleipulver in einer Menge von 3 g pro Tier und Tag über das Futter an experimentell mit S. Enteritidis infizierte Legehennen verabreicht und die Ergebnisse mit denen einer Kontrollgruppe ohne Volleipulver verglichen. Die gelegten Eier wurden getrennt nach Eischale, Eiklar und Eigelb auf den Infektionsstamm untersucht. Im Versuchskomplex C wurden die Adhäsivität und Invasivität verschiedener Salmonella-Stämme an bzw. in Epithelzellen bei An- bzw. Abwesenheit von Anti-S. Enteritids-Eigelb- bzw. -Serumantikörpern unter Verwendung einer permanenten Dünndarmepithelzelllinie von der Ratte (IEC-6) nach Inkubation durch Keimzählung überprüft. Die Ergebnisse in Versuchskomplex A zeigten, dass Eigelbantikörper nicht in der Lage sind, im Eigelb befindliche Salmonellen in ihrer Vermehrung zu hemmen oder gar abzutöten. Die Infektionsversuche im Versuchskomplex B ergaben, dass bei einer Infektionsdosis von 2x109 koloniebildenden Einheiten (kbE)/Tier die Salmonella-Kontaminationsrate der Eier durch das antikörperhaltige Eipulver nicht gesichert verändert wird. Dagegen konnte beim Einsatz einer solchen von 2x108 kbE/Tier eine Wirkung des antikörperhaltigen Eipulvers auf die Kontamination der einzelnen Eikompartimente nachgewiesen werden. Dabei wurden in der Versuchsgruppe mit 13,3 % gesichert weniger Eier als positiv detektiert als in der Kontrollgruppe mit 29,4 % (p &#8804; 0,001), wobei mindestens in einem der drei Kompartimente Eischale, Eiklar oder Eigelb Salmonellen nachweisbar waren. In der Versuchsgruppe wies jedes Salmonella-positive Ei auch Keime auf der Eischale auf. In der Kontrollgruppe waren mit 29,9 % signifikant mehr Eischalen positiv als in der Versuchsgruppe mit 13,3 % (p &#8804; 0,001). Beim Eiklar ließ die geringe Nachweisrate sowohl in der Versuchs- als auch in der Kontrollgruppe eine statistische Beurteilung des Eipulvereinflusses nicht zu. Aus dem Eidotter wurden in der Versuchsgruppe bei 0,6 % der Eier Salmonellen isoliert, während es in der Kontrollgruppe mit 2,4 % signifikant mehr waren (p &#8804; 0,05). Die Ergebnisse des Versuchskomplexes C zeigten, dass Volleiantikörper und Serumantikörper die Invasion von S. Enteritidis in intestinale Epithelzellen serovarspezifisch vermindern können. Insgesamt lassen die Ergebnisse die Schlussfolgerung zu, dass bei einer oralen Verabreichung von Volleipulver mit S. Enteritidis-spezifischen Antikörpern an Legehennen bei einer Infektionsbelastung von 2x108 kbE/Tier mit einer gesicherten Reduzierung des Vorkommens von Salmonellen im Nahrungsmittel Ei im Vergleich zu Tieren ohne Zugabe von Eipulver zu rechnen ist. / In vitro and in vivo studies on the efficiency of egg yolk antibodies against Salmonella Enteritidis Michael Gürtler Institute of Food Hygiene, Faculty of Veterinary Medicine, University of Leipzig Infection of humans by Salmonella are among the most important zoonotic diseases. Contaminated food, especially eggs and egg products, represents the highest risk. Still Salmonella Enteritidis (S. Enteritidis) dominates, a serovar that occurs especially in poultry flocks. A possibility to combat Salmonella infection is the passive immunisation of laying hens by oral administration of egg yolk antibodies. The application of antibody-containing full-egg powder against bacterial diseases was tested previously especially on calves and piglets. The suitability of administration of egg-yolk antibodies to protect laying-hens flocks against Salmonella was not tested previously. It was the aim of the study to investigate the influence of egg-yolk antibodies of S. Enteritidis in egg laying hens by in-vivo and in-vitro studies. In part A, we investigated the influence of anti-S. antibodies in egg yolk on the multiplication of Salmonella in vitro. In part B, the impact of oral application of anti-Salmonella full-egg antibodies on the in-vivo Salmonella-contamination rate of eggs layed by experimentally infected hens was investigated. In part C the ability of egg-yolk antibodies and serum antibodies were tested with regard to the rate of adhesion and invasion of Salmonella on or in epithelial cells of a permanent cell culture line (two major pathogenic mechanisms). In part A, laying hens were hyper-immunised parenterally by an inactivated S. Enteritidis strain to induce a high concentration of antibodies in the egg yolk. Egg yolk was than inoculated with two S. Enteritidis strains in three different contamination doses. Multiplication of Salmonella cells was detected by cell counts and calculated as generation time. In part B, laying hens were immunised passively with anti-Salmonella egg yolk antibodies (by feeding 3 g of full egg powder per day and per animal) and the effects were compared with a control group without full-egg powder. The inoculation strain of S. Enteritidis was detected on egg shell, in egg white and in egg yolk separately. In part C, a suspension of S. Enteritidis was added to a permanent duodenal epithelial cell line of the rat (IEC-6). After incubation the adhesivity of Salmonella strains and their ability to invade these cells in the presence or in the absence of anti-S. Enteritidis egg yolk and serum antibodies, respectively, was detected by cell count. The results of part A demonstrated that egg-yolk antibodies are not able to inhibit multiplication of Salmonella in egg yolk. Experiments of part B showed that the Salmonella contamination rate of eggs could not be influenced by antibody-containing egg powder at a Salmonella infection dose of 2x109 colony forming units (cfu)/animal, whereas the antibody-containing egg powder at an Salmonella infection dose of 2x108 cfu/animal proved effective (i.e. contamination rate of egg components was reduced). In the experimental group 13.3 % of the eggs were Salmonella positive, which was significantly different from 29.4 % in the control group (p &#8804; 0,001). At least in one of the three compartiments egg shell, egg white or egg yolk Salmonella was detectable. In the experimental group Salmonella could be isolated from the shell of every positive egg. The rate of Salmonella isolation from egg shells of the control group was significantly higher those in the experimental group (p &#8804; 0,001). With regard to egg white, the low number of Salmonella detections in both groups did not allow statistical evaluation of the effect of full-egg powder. From egg yolk of the experimental group in 0.6 % of the eggs Salmonella could be isolated which was significantly lower than the detection rate in the control group with 2.4 % (p &#8804; 0,05). The results of part C demonstrated that full-egg antibodies and serum antibodies can inhibit the serovar-specific invasion of S. Enteritidis into intestinal epithelial cells. As a whole, the results support the conclusion that oral administration of full-egg powder containing S. Enteritidis specific antibodies to egg-laying hens exposed to a administration of 2x108 cfu/animal reduce the occurrence of Salmonella in the egg compared to eggs of animals without egg powder administration.

No description available.

Das Flagellum von Salmonella enterica ist eine komplexe molekulare Nanomaschine, die zur Fortbewegung verwendet wird. Die Synthese erfordert die Sekretion extrazellulärer Bausteine durch die Zellhülle. Der Substratexport erfolgt durch ein hochkonserviertes Typ-III-Sekretionssystem. Der Kern des fT3SS ist eine komplexe Proteinsekretionsmaschine, bestehend aus den Proteinen FliPQR und FlhBA. Ziel dieser Arbeit war die molekularen Mechanismen, die eine korrekte Funktion gewährleisten, tiefergehend zu erforschen. Im ersten Kapitel wurden die molekulare Mechanismen der Qualitätskontrolle während der Synthese des fT3SS untersucht. Es wurde kürzlich gezeigt, dass die korrekte Synthese durch das fT3SS-spezifischen Chaperon FliO gewährleistet wird. Ziel war es, den molekularen Mechanismus, wie FliO an diesem Prozess beteiligt ist, aufzuklären. Die Ergebnisse zeigten, dass mehrere Aminosäuren von FliO während der Assemblierung mit FliP interagieren. Des Weiteren wurde die Relevanz des spaltbaren Signalpeptids am N-Terminus von FliP untersucht. Diese Studie zeigt, dass die Anwesenheit des Signalpeptids und seine korrekte Spaltung entscheidend, aber nicht unerlässlich für die Funktion der Flagellen sind. Das fT3SS ist in der Lage Proteine mit einer bemerkenswerten Geschwindigkeit von mehreren tausend Aminosäuren pro Sekunde zu sekretieren. Das zweite Kapitel konzentrierte sich darauf, wie das fT3SS Proteine mit hoher Geschwindigkeit sekretiert, während das Austreten kleiner Moleküle verhindert wird. Unsere Mutationsanalysen zeigten, dass eine Methioninschleife in FliP, eine sperrige Plug-Domäne in FliR und intermolekulare Salzbrücken zwischen FliQ-Untereinheiten zusammenarbeiten, um die Integrität der Membran aufrechtzuerhalten. Diese Arbeit liefert neue Einblicke in die Synthese des fT3SS Kerns und die Regulation der Substratsekretion. Beide Prozesse werden an mehreren Stellen streng kontrolliert, um eine korrekte Funktion des Flagellums sicherzustellen. / The flagellum of Salmonella enterica is a sophisticated molecular nanomachine, which is used for locomotion. Flagella synthesis requires the translocation of extracellular subunits across the cell envelop, which is mediated by a highly conserved type-III secretion system (fT3SS). The core fT3SS is a complex protein secretion machine consisting of the proteins FliPQR and FlhBA. Productive assembly is crucial for flagella function. The molecular mechanisms which ensure correct function of the fT3SS remain poorly understood. In this thesis, we aimed to gain a profound insight into the molecular mechanisms of fT3SS core assembly and function. The first chapter investigated the molecular mechanisms underlying the quality control during the assembly of the fT3SS. It was recently shown that productive assembly of the core fT3SS relies on the flagella-specific chaperone FliO. We aimed to elucidate the molecular mechanism of how FliO facilitates this process. Our results demonstrated, that several residues of FliO are interacting with FliP during the assembly process. Furthermore, we aimed to identify the relevance of the cleavable signal peptide at the N-terminus of FliP. This study showed, that the presence of the signal peptide and its correct cleavage are crucial but not essential for flagella function. The fT3SS is able to secrete proteins with a remarkable speed of several thousand amino acids per second. The second chapter focused on how the fT3SS secretes proteins at high speed while preventing the leakage of small molecules. Our mutational analyses demonstrated that a methionine loop in FliP, a bulky plug domain in FliR and intermolecular salt bridges between FliQ subunits are acting cooperatively to maintain the membrane barrier. Overall, this work provides new insights into the assembly process of the fT3SS core and the regulation of substrate secretion. Both processes are tightly controlled at multiple stages to ensure the proper functioning of the flagellum.

No description available.

No description available.

In the twenty-first century, advances in synthetic biology and molecular tools to implement programmable behavior into microbes have fueled significant efforts to develop microbial-based therapeutics. Bacteria and viruses have been explored independently for their ability to replicate and induce cytotoxic effects in cancer cells selectively. This dissertation aims to co-opt the anti-tumor capabilities of gram-negative Salmonella enterica subspecies enterica serotype Typhimurium (referred to as Salmonella typhimurium moving forward) and picornaviruses (small RNA viruses with positive sense genomes) to develop a potent, single bacterial-viral consortium- based system to treat solid tumors.I first describe our efforts to co-opt S. typhimurium’s natural internalization into hosts and intracellular space-sensing to deliver self-amplifying picornaviral RNA. Protein effectors that promote intracellular survival of S. typhimurium within the Salmonella-Containing-Vacuole (SCV) are transcribed by Salmonella Pathogenicity Island-2 (SPI-2) promoters, which turn on after sensing the intracellular pH, ion concentrations, and oxidative stressors. These effectors are then translocated into the host’s cytoplasm by a needle apparatus that connects the SCV and cytoplasm, which is also transcribed by SPI-2 promoters. By using the SPI-2 promoter PsseA to drive the expression of fluorescent reporters and membrane-disrupting proteins (eukaryotic and prokaryotic), efficient escape of Salmonella-produced proteins into tumor-host cells was established. RNA delivery into host cells was also made possible by a secondary SPI-2 promoter, PsseJ, which transcribes RNA polymerase T7 (T7), which then transcribes a T7-promoter-driven Poliovirus replicon or full-length Senecavirus A (SVA). Inoculation of this engineered S. typhimurium strain on a panel of cancer cell lines identified the system’s ability to deliver viral replicons and full-length viruses in a small cell cancer cell line, H446. In a murine model, S. typhimurium delivery of SVA was then shown to clear xenografted H446 tumors. Motivated by the possibility of delivering other picornaviral species with similar anti-tumor properties, but documented healthy tissue cytotoxicity, S. typhimurium was further engineered to control SVA viral spread. By driving Tobacco Etch Virus (TEV) protease expression via a second SseA promoter, and replacing a natural cleavage site on SVA with the TEV-cleavage domain, we demonstrate TEV-dependent SVA spread in H446 cells. I conclude with efforts on engineering TEV-dependent-SVA transgene expression to confer greater antitumor properties. Interferon-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to attenuate H446 growth in vitro. Expression of interferon-gamma off SVA would produce a direct selective pressure against viral replication and virion production. However, a fusion of human GM-CSF to Nano-Luciferase protein on the TEV-dependent SVA genome maintained luminescent signals, GM-CSF activity, and TEV-dependent spread, providing a framework to survey anti-tumor properties of SVA-transgenes. Furthermore, I address our development of syngeneic models for Salmonella-mediated delivery of SVA, an important step towards clinical applications of the system as immunocompetent models more closely correlate to immunocompetent patient populations. SVA’s efficient entry and replication in neuroendocrine-derived tissue identified murine neuroblastoma N1E-115 cells as a suitable cell line for SVA cytotoxicity studies. However, the ability of these cells to support bacterial-viral superinfections is unknown. Here, we show that Salmonella-mediated launch of SVA leads to viral spread that can attenuate heterologous hind flank tumor growth and improve their survival along with mice engrafted with orthotopic intracranial brain tumors.