Hohenheim, Univ., Diss., 2007.

Würzburg, Univ., Diss., 2008. / Zsfassung in engl. Sprache.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Würzburg, Univ., Diss., 2001.

Listeria monocytogenes is a rod-shaped Gram positive bacterium. It generally exist ubiquitously in nature, where it lives as a saprophyte. Occasionally it however enters the food chain, from where it can be ingested by humans and cause gastro-intestinal distress. In immunocompetent individuals L. monocytogenes is generally cleared within a couple of weeks, but in immunocompromised patients it can progress to listeriosis, a potentially life-threatening infection in the central nervous system. If the infected individual is pregnant, the bacteria can cross the placental barrier and infect the fetus, possibly leading to spontaneous abortion. The infectivity of L. monocytogenes requires a certain set of genes, and the majority of them is dependent on the transcriptional regulator PrfA. The expression and activity of PrfA is controlled at several levels, and has traditionally been viewed to be active at 37 °C (virulence conditions) where it bind as a homodimer to a “PrfA-box” and induces the expression of the downstream gene. One of these genes is ActA, which enables intracellular movement by recruiting an actin polymerizing protein complex. When studying the effects of a blue light receptor we surprisingly found an effect of ActA at non-virulent conditions, where it is required for the bacteria to properly react to light exposure. To further study the PrfA regulon we tested deletion mutants of several PrfA-regulated virulence genes in chicken embryo infection studies. Based on these studies we could conclude that the chicken embryo model is a viable complement to traditional murine models, especially when investigating non-traditional internalin pathogenicity pathways. We have also studied the effects of small molecule virulence inhibitors that, by acting on PrfA, can inhibit L. monocytogenes infectivity in cell cultures with concentrations in the low micro-molar range.

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-04-17T13:58:12Z No. of bitstreams: 2 Tese Ana Paula da Costa.pdf: 11703201 bytes, checksum: 21beb28c4cce405340195e0c8ba78a09 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-17T13:58:12Z (GMT). No. of bitstreams: 2 Tese Ana Paula da Costa.pdf: 11703201 bytes, checksum: 21beb28c4cce405340195e0c8ba78a09 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-28 / FACEPE / Listeria monocytogenes é um bacilo gram-positivo, intracelular facultativo, transmitido por alimentos, que pode causar doença, a listeriose, em indivíduos susceptíveis. Embora rara, a listeriose tem grande importância para a saúde pública pela sua alta letalidade. Este trabalho teve como objetivo, identificar marcadores moleculares para utilização no desenvolvimento de nova técnica de diagnóstico molecular da listeriose. Os estudos envolveram sorotipagem, tipagem molecular pela amplificação das sequências 23S, 16S-23S e RAPD, pesquisa de plasmídios e de genes de virulência (inlA, inlB, inlC, inlJ, hly, iap, plcA, actA) em 135 cepas de L. monocytogenes de casos humanos e alimentos isoladas no Brasil no período de 1975 a 2001; infecção experimental em camundongos com cepas portando ausência ou alterações em genes de virulência; e a caracterização de isolados de um caso de endocardite atendido numa unidade de emergência cardiológica em Recife, PE, em 2010. Nenhuma relação foi encontrada entre a origem das cepas, sorotipos, perfis genéticos, conteúdo plasmidial e distribuição dos genes de virulência. Os ensaios de infecção experimental em camundongos também não permitiram estabelecer uma relação entre a presença dos marcadores de virulência considerados no estudo e as respostas dos animais inoculados. Seis isolados de hemocultura de um caso de endocardite com identificação presuntiva de Listeria spp. foram classificados como L. monocytogenes sorotipo 4b pela amplificação por PCR dos genes 23S, lmo2243 e ORF2210 específicos do gênero Listeria, espécie L. monocytogenes e sorotipo b respectivamente. Todos os genes de virulência pesquisados foram detectados por PCR nas amostras e a análise do perfil de amplificação da sequência 16S-23S revelou que os seis isolados são uma mesma cepa. Embora esses estudos não tenham revelado nenhum marcador molecular de patogenicidade, a alta frequência de genes de virulência observada entre cepas dos sorotipos mais patogênicos (1/2a, 1/2b, 4b) evidencia o potencial patogênico nas cepas brasileiras de L. monocytogenes e a necessidade de incrementar a vigilância e diagnóstico dessa bactéria. Considerando a prevalência dos sorotipos 1/2a, 4b nas amostras clinicas e dos sorotipos 1/2a, 1/2b, 4b em alimentos, desenvolvemos e padronizamos um procedimento baseado em LAMP (loop-mediated isothermal amplification) para identificação desses sorotipos. A técnica é rápida e de fácil operacionalização, utiliza um bloco de aquecimento ou banho-maria e o produto da reação pode ser visualizado a olho nu mediante adição de reagentes fluorescentes. O procedimento padronizado mostrou-se sensível, capaz de detectar 100pg de DNA ou 104 UFC, e espécie - específica, capaz de diferenciar L. monocytogenes de espécies intimamente relacionadas geneticamente. Outros sorotipos intimamente relacionados (3a, 3b, 4e e 4d) foram amplificados sem detrimento da técnica porque são raramente encontrados em amostras humanas e animais. A técnica LAMP se apresenta como uma alternativa fácil e rápida para diagnóstico e tipagem de L. monocytogenes especialmente para os programas de vigilância e investigação epidemiológica.

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-22T23:06:06Z (GMT). No. of bitstreams: 1 Kabuki_DirceYorika_M.pdf: 13196927 bytes, checksum: cd5b73913adb45b689c065ec84b7f5e9 (MD5) Previous issue date: 1997 / Resumo: Atualmente a L. monocytogenes é considerada um patógeno de importância em alimentos, pois vários surtos e casos de listeriose associados ao consumo de alimentos têm sido relatados, incluindo os produtos de frango. O aumento da produção brasileira de carnes de aves, a redução de preço no mercado interno e conseqüente aumento do consumo e a falta de métodos oficiais para a quantificação de L. monocytogenes em alimentos, principalmente os in natura, levaram ao desenvolvimento deste projeto numa tentativa de quantificá-la para que limites máximos de tolerância possam ser propostos. Amostras resfriadas de peito e de frango inteiro obtidas no comércio varejista, foram submetidas a quantificação de Listeria spp pelo método do Número Mais Provável (NMP) utilizando-se: a) caldo Fraser modificado (MdFB) contendo 20 mg de acriflavina/L a 35°C/48h; b) caldo de enriquecimento para Listeria (LEB) (USDA) a 30°C/48h e c) caldo LEB a 30°C/24h seguido do caldo Fraser modificado (MFB) (USDA) a 35°C/24-48h. No isolamento foram utilizados os ágares cloreto de lítio feniletanol moxalactam (LPM) e Oxford modificado (MOX) e na identificação as provas bioquímicas padrão ou o kit API-Listeria. Os resultados revelaram: i) a viabilidade do emprego do meio MdFB na técnica do NMP; ii) valores de NMP para Listeria sp e L. monocytogenes entre <0,3 a 93/g para peitos e entre <9 a 2,8x103/carcaça ou <0,005 a 1,943/g de frango; iii) predomínio de L. innocua, L. monocytogenes, L. welshimeri e L. seeligeri. Em estudo paralelo com as mesmas amostras, verificou-se ocorrência elevada de Listeria sp e L. monocytogenes, com índices de 96,7% e 90,0% respectivamente e pequena diferença entre carcaças e peitos de frango. O ágar LPM apresentou desempenho melhor que o MOX no isolamento de L. monocytogenes. Em outro estudo, empregando-se a técnica do NMP proposta, verificou-se a ineficiência de hipoclorito de sódio e de fosfato trissódico na redução de L. monocytogenes em coxas de frango artificialmente contaminadas; os níveis de redução obtidos foram ? 0,42 ciclos log, os quais não foram considerados estatisticamente significantes / Abstract: L. monocytogenes is currently considered to be an important food pathogen, since various outbreaks and cases of listeriosis have been related to the consumption of foods, including some chicken products. The increase in the production of fowl meats in Brazil, the reduction of the price on the internal market and consequent increase in consumption by the population and the lack of official methods for the quantification of L. monocytogenes in foods, principally in raw foods, are the factors which led to the development of this project, aimed at quantifying the problem and subsequently proposing maximum tolerance limits. Thus refrigerated samples of chicken breast and whole chickens, ali obtained on the retail market, were quantified for Listeria spp by the Most Probable Number (MPN) method, using a) modified Fraser broth (MdFB) containing 20 mg acriflavine/L at 35°C/48h; b) Listeria enrichment broth (LEB) (USDA) at 30°C/48h and c) LEB broth at 30°C/24h followed by modified Fraser broth (MFB) (USDA) at 35°C/24­48h. For the isolation procedure, lithium chloride phenylethanol moxalactam agar (LPM) and modified Oxford agar (MOX) were used, and the standard biochemical test or API­ Listeria kit used for identification. The results showed the following: i) the viability of using the medium MdFB in the MPN technique; ii) MPN values for Listeria spp and L. monocytogenes between <0.3 and 93/g for breasts and between <9 and 2.8x103/carcass or <0.005 to 1.943/g chicken and iii) predominance of L. innocuti, L. monocytogenes, L. welshimeri and L. seeligeri. In a parallel study with the same samples, an elevated occurrence of Listeria spp and L. monocytogenes was determined, with indices of 96.7% and 90.0% respectively, the difference between the carcasses and breasts being relatively small. LPM agar was shown to be a better isolation media for L. monocytogenes than MOX. In another study using the proposed MPN technique, the inefficiency of sodium hypochlorite and of trisodium phosphate in the reduction of L. monocytogenes in artificially contaminated chicken legs was shown, the reduction levels being ?0.42 log cycles, and not therefore statistically significant / Mestrado / Mestre em Tecnologia de Alimentos

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.

Persistence of Listeria monocytogenes in food processing plants is a huge health and economic burden. Biofilms are considered to be one of the major mechanisms by which this pathogen persists within these environments. Studies so far have mostly used optimal growth conditions in their investigations which may not provide a realistic understanding of the biofilm forming abilities of L. monocytogenes in food processing plants. Therefore the aim of this study was to 1) establish a model (12 ºC, Beef Broth) that closely relates to the food processing environment 2) screen 66 isolates of L. monocytogenes from food and clinical sources and determine their biofilm forming phenotypes (non-, weak, moderate and strong formers) and 3) analyze the correlation between biofilm formation phenotypes and biofilm associated genes detected using polymerase chain reaction (PCR) and Basic Local Alignment Search Tool (BLAST) for whole genome sequences. Biofilm formation established at 12 ºC in Beef Broth was the most consistent and quantifiable at day 9 of incubation. Subsequently, 66 isolates were screened using this model, resulting in 60 isolates being identified as strong biofilm formers, 5 isolates as moderate biofilm formers and 1 isolate as a weak biofilm former. Twenty biofilm associated genes were analyzed using PCR in 27 representative isolates. Out of the 20 genes, at least 17 of them were detected in all the tested isolates. Out of the 106 biofilm associated genes analyzed using BLAST, all the isolates were found to show the presence of at least 92 genes. In conclusion, there was no obvious correlation between the presence/absence of the genes selected for analysis and the ability to form biofilms using approaches performed in this study. However, the model established in the study will be useful in further analysis (transcription and translation studies) of genetic markers responsible for biofilm formation of L. monocytogenes under food processing conditions.

Mutations in the Leucine-rich repeat kinase 2 (Lrrk2) gene are associated with familial and sporadic cases of Parkinson’s disease but are also found in inflammatory-related disorders such as Crohn’s disease, systemic lupus erythematosus, tuberculosis and leprosy. There is also evidence that LRRK2 is highly expressed in immune cells, particularly in macrophages, and has been functionally linked to pathways pertinent to immune cell function such as modulating the course of infections, cytokine release, autophagy and phagocytosis. Indeed, G2019S mutation in Lrrk2 is the most common mutation in Parkinson’s disease. Accordingly, we hypothesized that G2019S mutation in Lrrk2 might enhance the activation of the innate immune system. We tested our hypothesis by performing challenge experiments in a mouse model of Listeria monocytogenes, and by measuring the activation of bone marrow derived macrophages (BMDMs) following in vitro infection with the bacterium. We found that Lrrk2G2019S mutant mice controlled L. monocytogenes better than WT mice. The mechanism behind the better control of L. monocytogenes by the G2019S mutation of Lrrk2 was investigated in BMDMs following in vitro infection with L. monocytogenes. Interestingly, we found that Lrrk2G2019S mutation enhances the production of TNF-α, IL-1β and IL-10 by infected BMDMs. The impact on TNF-α and IL-1β was specifically due to the G2019S mutation of Lrrk2 since there was no impact on the expression of these cytokines in Lrrk2 knockout macrophages. Western blotting experiments revealed that the G2019S mutation of Lrrk2 enhances MAPK signaling (TAK1, p38 and ERK). Modulation of the expression of the pro-inflammatory cytokines, TNF-α and IL-1β by G2019S mutation of Lrrk2 occurred via p38 MAPK activation. The impact on IL-10 expression occurred through increased ERK activation by the G2019S mutation of Lrrk2. We did not observe any impact of G2019S mutation of Lrrk2 on the activation of NF-κB and JNK MAPK pathways. Increased expression of IL-1β by G2019S mutation of Lrrk2 revealed increased inflammasome signaling. Inflammasome signaling in response to L. monocytogenes was mainly mediated by the AIM2- and partly by NLRP3- inflammasome and was dependent on activation of caspase-1. We found that Lrrk2G2019S mutation enhanced the expression of NLRP3 and caspase-1. Finally, we found that the expression of reactive oxygen species (ROS) following infection with L. monocytogenes was augmented by G2019S mutation of Lrrk2, and this can be an important mechanism that promotes the enhanced clearance of the bacterium in vivo. Overall, these results present new insights into the signaling mechanisms through which the G2019S mutation of Lrrk2 augments innate immune response which leads to better control of infection.