Thèse d'exercice : Pharmacie : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

No description available.

Memoria para optar al Titulo Profesional de Médico Veterinario / En este estudio se valoró el efecto letal de la técnica de cocción al vacío (sous-vide) sobre Listeria monocytogenes (Scott A y CECT 4031) inoculada en carne de cerdo, previamente refrigerada (4ºC durante 24 h) o congelada (-20ºC durante 15 días), y en la microbiota aerobia mesófila contaminante. De igual forma se evaluó la posibilidad de recuperación y multiplicación de los microorganismos durante su posterior conservación (4 y 8ºC). Las muestras de lomo de cerdo (Longissimus dorsi) fueron tratadas en horno mixto de convección-vapor a 60ºC durante 30, 60 y 90 min. No se observaron diferencias significativas en las letalidades obtenidas en función de cada tratamiento, tanto para los microorganismos aerobios mesófilos como para Listeria monocytogenes, evidenciándose en esta última una reducción de 6 log10 UFC/g para ambas cepas. La temperatura de almacenamiento tuvo un efecto significativo en la recuperación de los microorganismos aerobios mesófilos, disminuyendo su vida útil en las muestras conservadas a 8ºC. Al comparar la materia prima inicialmente refrigerada con la congelada, se observaron diferencias significativas únicamente en las muestras tratadas durante 30 min y almacenadas a 8ºC en la cepa Scott A. La cepa CECT 4031 no alcanzó los 2 log10 UFC/g ningún día de análisis, tanto para las muestras refrigeradas como congeladas. Lo mismo ocurrió en la cepa Scott A para las muestras congeladas, sin embargo, las muestras inicialmente refrigeradas y tratadas durante 30 min, alcanzaron dicho recuento a partir del día 5, observándose un efecto significativo de la temperatura de conservación a 8ºC, reduciendo de esta forma su vida comercial. Para las muestras tratadas a 60°C durante 60 o 90 min, el indicador para determinar la vida útil es el recuento total de aerobios mesófilos, mientras que para las muestras tratadas a 60°C durante 30 min, el microorganismo indicador es Listeria monocytogenes (cepa Scott A), especialmente cuando la temperatura de conservación no se mantiene escrupulosamente inferior o igual a 4ºC / proyecto “Estudio de vida comercial y efecto de la letalidad de la técnica de cocción sous-vide”, financiado y realizado por la Universidad Autónoma de Barcelona, Barcelona, España

In the present study, a new gene cluster of Listeria monocytogenes EGD containing three internalin genes was identified and characterized. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the class of large, surface-bound internalins. Each of these proteins contains a signal peptide, two regions of repeats (Leucine-rich repeats and B repeats), an inter-repeat region and a putative cell wall anchor sequence containing the sorting motiv LPXTG. PCR analysis revealed the presence of the inlGHE gene cluster in most L. monocytogenes serotypes. A similar gene cluster termed inlC2DE localised to the same position on the chromosome was described in a different L. monocytogenes EGD isolate. Sequence comparison of the two clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and a 5'-portion of inlD. The genes inlG, inlH and inlE seem to be transcribed extracellularly and independent of PrfA. To study the function of the inlGHE gene cluster several in-frame deletion mutants were constructed which lack the genes of the inlGHE cluster individually or in combination with other inl genes. When tested in the mouse model, the inlGHE mutant showed a significant reduction of bacterial counts in liver and spleen in comparison to the wild type strain, indicating that the inlGHE gene cluster plays an important role in virulence of L. monocytogenes. The ability of this mutant to invade non-phagocytic cells in vitro was however two- to three-fold higher than that of the parental strain. To examine whether deletion of the single genes from the cluster has the same stimulatory effect on invasiveness as deletion of the complete gene cluster, the single in-frame deletion mutants inlG, inlH and inlE were constructed. These mutants were subsequently reverted to the wild type by introducing a copy of the corresponding intact gene into the chromosome by homologous recombination using knock-in plasmids. To determine a putative contribution of InlG, InlH and InlE in combination with other internalins to the entry of L. monocytogenes into mammalian cells, the combination mutants inlA/GHE, inlB/GHE, inlC/GHE, inlA/B/GHE, inlB/C/GHE, inlA/C and inlA/C/GHE were constructed. Transcription of the genes inlA, inlB and inlC in these mutants was studied by RT-PCR. Deletion of inlGHE enhances transcription of inlA and inlB, but not of inlC. This enhancement is not transient but can be observed at different time-points of the bacterial growth curve. Deletion of inlA also increases transcription of inlB and vice-versa. In contrast, the amounts of inlA and inlB transcripts in the single deletion mutants inlG, inlH and inlE were similar to those from the wild type. / In der vorliegenden Arbeit wurde ein neues Gencluster von Listeria monocytogenes EGD mit drei Internalingenen identifiziert und charakterisiert. Diese als inlG, inlH und inlE bezeichneten Gene codieren für Proteine mit 490, 548 bzw. 499 Aminosäuren, die zur Klasse der großen, oberflächengebundenen Internaline gehören. Jedes dieser Proteine enthält ein Signalpeptid, zwei Repeat-Regionen (Leucin-reiche Repeats und B-Repeats), eine Inter-Repeat-Region, und eine mögliche Zellwandankersequenz mit dem Motiv LPXTG. PCR-Analyse zeigte das Vorkommen des inlGHE-Genclusters in den meisten L. monocytogenes-Serotypen. Ein ähnliches, als inlC2DE bezeichnetes Gencluster wurde in der gleichen Position auf dem Chromosom eines anderen L. monocytogenes EGD Isolats beschrieben. Ein Sequenzvergleich beider Clusters zeigte, dass inlG ein neues Internalingen ist, während inlH durch eine in-frame-Deletion vom 3'-Ende von inlC2 und einem 5'-Teil von inlD entstanden ist. Die Gene inlG, inlH und inlE werden vorwiegend extrazellulär und PrfA-unabhängig transkribiert. Um die Funktion des inlGHE-Genclusters zu untersuchen, wurden verschiedene in-frame-Deletionsmutanten hergestellt, aus denen die Gene des inlGHE-Clusters entweder einzeln oder in Kombination mit anderen Internalingenen deletiert wurden. Im Maumodell zeigte eine inlGHE-Mutante nach oraler Infektion eine signifikante Reduktion der Bakterienzahl in der Leber und Milz, die auf eine wichtige Rolle des inlGHE-Genclusters in der Virulenz von L. monocytogenes hindeutet. Die Fähigkeit dieser Mutante, in nicht-phagocytische Zellen in vitro einzudringen, ist zwei bis dreifach höher als die des Wildtyp-Stammes. Um zu untersuchen, ob die Deletion von Einzelgenen des inlGHE-Genclusters einen ähnlichen stimulatorischen Effekt auf die Invasivität ausübt wie die Deletion des kompletten Genclusters, wurden die Einzeldeletionsmutanten inlG, inlH and inlE hergestellt. Diese Mutanten wurden anschließend zum Wildtyp revertiert, indem eine Kopie des entsprechenden intakten Gens durch homologe Rekombination ins Chromosom mit Hilfe von Knock-in-Plasmiden eingeführt wurde. Um eine mögliche Rolle von InlG, InlH und InlE in Verbindung mit anderen Internalinen bei der Aufnahme von L. monocytogenes in Säugerzellen zu untersuchen, wurden die Mutanten inlA/GHE, inlB/GHE, inlC/GHE, inlA/B/GHE, inlB/C/GHE, inlA/C and inlA/C/GHE hergestellt. Die Transkription der Gene inlA, inlB and inlC in diesen Mutanten wurde durch RT-PCR untersucht. Deletion von inlGHE erhöht die Transkription von inlA und inlB, aber nicht die von inlC. Diese Erhöhung ist nicht vorübergehend sondern kann zu verschiedenen Zeitpunkten der Wachstumskurve beobachtet werden. Deletion von inlA verstärkt ebenfalls die Transcription von inlB und umgekehrt. Im Gegensatz dazu waren die Mengen an inlA- und inlB-Transkripten in den Einzeldeletionsmutanten inlG, inlH and inlE ähnlich wie die des Wildtyps.

Virulenz-attenuierte Bakterien sind geeignete Vektoren für den Transport von Vakzine-DNA in das Zytosol von Antigen-präsentierenden Zellen ("DNA delivery"). In dieser Arbeit wurde dazu das intrazelluläre Bakterium Listeria monocytogenes verwendet, welches sich im Zytosol von Zellen vermehrt und fortbewegt. Ausgestattet mit einer intrazellulären Lysis-Kassette kann Listeria in vitro effektiv Plasmid-DNA in das Zytosol verschiedener Zelltypen freisetzen. Zur Virulenz-Attenuierung wurde das Gen iap im Chromosom des Bakteriums deletiert. Der daraus resultierte Stamm, in Folgenden als &#61508;iap bezeichnet, erwies sich als hoch attenuiert im Modell der murinen Listeriose. Diese Attenuation konnte auf einen Defekt in der Beweglichkeit der Bakterien innerhalb von Wirtszellen zurückgeführt werden, da sich bei diesem Stamm das Protein ActA, das essentiell für die Aktin-basierte Motilität von L. monocytogenes ist, fehlerhaft auf der Oberfläche der Bakterien anordnet. Zusätzlich konnte demonstriert werden, dass &#61508;iap in der Zellteilung beeinträchtigt ist und deshalb eine veränderte Morphologie aufweist. Im Rahmen dieser Arbeit wurde ein so genanntes "Balanced-lethal" System etabliert. Dazu wurde das essentielle Gens trpS im Chromosom deletiert, während gleichzeitig eine trpS-Expressions-Kassette auf einem Vakzine-Plasmid inseriert wurde. Dieses System gewährleistet, dass das Trägerbakterium dieses Plasmid weder in vitro noch in vivo verliert. Dies ist besonders wichtig im Hinblick auf eine bakteriolytische Lysis-Kassette, welche ebenfalls auf diesem Plasmid kodiert ist. Es wurden verschiedene Lysis-Kassetten, die alle aus einem Listeria-spezifischen Phagenlysin und einem vorangestellten zytosolischen listeriellen Promotor zusammengesetzt waren, miteinander verglichen. Dabei wurde beobachtet, dass die für die Übertragung von Plasmid-DNA in das Zytosol von Wirtszellen wirksamste Phagenlysin-Kassette (PactA-ply118) die Bakterien in vitro nur partial abtötet, während sie in vivo zu einer besonders hohen Attenuation der Bakterien führt. Unter Verwendung dieses "DNA delivery" Systems wurden Mäuse oral mit Listerien infiziert, die ein DNA-Vakzine-Plasmid zur Expression des Leishmania Antigens KMP-11 trugen. Dabei konnte bei 27 % aller Tiere, die zweimal mit diesen Listerien infiziert worden sind, eine KMP-11 spezifische, proliferative Immunantwort gemessen werden. Listerien, die einen Defekt in ihrer Motilität besitzen (delta-iap, delta-actA), erwiesen sich darin beeinträchtigt, Plasmid-DNA im Zytosol von Zellen freizusetzen. Anhand dieser Stämme konnte gezeigt werden, dass die Fähigkeit von L. monocytogenes, sich innerhalb von Zellen zu bewegen und in benachbarte Zellen einzudringen eine wichtige Voraussetzung für einen effizienten Transfer von Plasmid-DNA in vitro darstellt. / Virulence-attenuated bacteria are useful carriers to introduce a DNA vaccine into antigen presenting cells (DNA delivery). To this end, the intracellular bacterium Listeria monocytogenes was used in this work, which is able to replicate and spread inside host cells. Hence Listeriae are able to efficiently release plasmid DNA within the cytosol in vitro when they are provided with a cytosolic lysis cassette. The expression of the antigen by the cell leads to the presentation of antigenic epitopes on the cell's major histocompatibilty complex (MHC) class I molecules, due to the antigen being endogenous. This stimulates the activation of CD8+ T cells which are important for clearance of tumours, parasites and virus infected cells. In order to create a virulence-attenuated carrier strain the gene iap was deleted in the chromosome of the bacterium. The resulting strain, designated as &#61508;iap, was shown to be highly attenuated in mice. This was due to a defect of the intracellular motility since ActA, a protein which is necessary for actin-based motility of Listeria, was localised incorrectly at the bacterial surface. Additionally, it was demonstrated that &#61508;iap is impaired in cell division which leads to an altered cell morphology. In this work a so-called balanced-lethal plasmid system was established. The essential gene trpS was deleted from the chromosome of L. monocytogenes and a trpS transcription unit was inserted in a vaccine DNA plasmid thus ensuring that no plasmid loss happens in vitro and also within the host organism. This is in particular important in terms of a bacteriolytic lysis cassette which is also encoded by the plasmid. Different lysis cassettes were tested consisting of a Listeria-specific phage lysin and an intracellular promoter of Listeria. The cassette PactA-ply118 was found the be most effective due to its DNA delivery capacity but it mediates only a partial lysis of the intracellular bacteria. However, this cassette leads to a high attenuation of Listeria in mice. Using this DNA delivery system mice were orally infected with Listeria harbouring a KMP-11 expression plasmid. 27 % of animals infected twice exhibited a specific proliferative response to the leismanial antigen KMP-11. Listeriae with a defect in their spreading capacity (delta-iap, delta-actA) were impaired in the cytosolic release of plasmid DNA. With these strains it was demonstrated, that spreading is an important prerequisite for L. monocytogenes to be an efficient DNA delivery carrier in vitro.

Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.

Thèse de doctorat : Procédés biotechnologiques et alimentaires : Vandoeuvre-les-Nancy, INPL : 2006. / Titre provenant de l'écran-titre. Bibliogr.