Дисертації з теми "Escherichia-Shigella"

Escherichia coli enteroinvasora (EIEC) possuem características bioquímicas e genéticas semelhantes às de Shigella, porém para que ocorra um processo infeccioso são necessárias 102 células de Shigella em relação a 106 células de EIEC. A patogenicidade de Shigella e EIEC se dá pela presença de um plasmídio de virulência denominado pInv, que contêm os genes necessários para a invasão e disseminação bacteriana nas células do hospedeiro. Estudos anteriores relataram que os genes ipaA, ipaB, ipaC e ipaD de EIEC e Shigella não possuem diferenças genéticas que possam explicar sua diferença de patogenicidade. No presente trabalho, foram avaliados os níveis transcricionais dos genes envolvidos na invasão e disseminação dessas bactérias. Pela técnica de RT-PCR semi-quantitativo, pode-se observar diferenças nos níveis de transcrição para a maioria dos genes de virulência selecionados, quando as bactérias estavam em contato com os macrófagos. Porém, sem o contato com estas células, o nível de transcrição dos genes foi o mesmo entre as espécies, com exceção do gene ipaD. Foi verificada que a transcrição deste gene em contato com macrófago é praticamente a mesma entre as bactérias, enquanto que na ausência de macrófagos, o nível de transcrição é bem menor em EIEC, quando comparado no mesmo intervalo de tempo. Após estes resultados foram selecionados os principais genes para estudo por PCR em tempo real. Foi possível observar que o nível de transcrição de EIEC é menor, em relação a Shigella. Mais ainda, a análise dos genes dentro do mesmo operon mostrou uma cinética de transcrição distinta do icsB em relação a outros genes do operon das ipas. Os resultados obtidos sugerem que esta diferença de transcrição possa estar relacionada com a virulência mais branda em EIEC. Ainda mais, Os genes pertencentes ao operon icsB-ipaCB parecem ser regulados de forma distinta. Além disso, a transcrição de ipaD em EIEC, provavelmente, depende de uma via de sinalização distinta de Shigella flexneri. Diante desses resultados, novos estudos estão sendo propostos em nosso laboratório para melhor compreensão do mecanismo de virulência desse enteropatógeno.
Enteroinvasive Escherichia coli (EIEC) serotypes described so far share antigenic, biochemical, genetic and pathogenetic properties with Shigella sp. However, in order for an infectious process to occur, an inoculum of 102 Shigella cells is needed in contrast to as much as 106 EIEC cells. The characteristic ability of S. flexneri and EIEC to enter epithelial cells, multiply intracellularly and spread from cell to cell is uniquely encoded by their 220-kb virulence plasmid. Previous studies realized in our laboratory showed that the genes ipaA, ipaB, ipaC and ipaD do not possess molecular alterations in the nucleotides sequences that can explain the difference in the pathogenicity between EIEC and Shigella spp. In the present work, the transcription levels of the bacterial genes involved in the invasion and escape from host cells were evaluated. Through reverse transcription-polymerase chain reaction (RT-PCR) analysis, differences in the transcription levels for the majority selected virulence genes could be observed when bacteria were in contact with the macrophages. However, without the contact with those cells, the transcription levels of the genes were the same between both bacteria species, with the exception of ipaD. When the bacteria is in contact with macrophages, the transcription of ipaD is the same in both species whereas in the absence of macrophages, the transcription level is lower in EIEC than Shigella, when compared in the same period of time. Those results provided the selection of the genes for the real time PCR analysis. In general, the EIEC transcription levels genes are lower than Shigella. More still, the icsB showed a distinct kinetic of transcription from the others genes in the same operon. All results suggest that the lower pathogenicity due to EIEC can partially have to the differences of the virulence genes transcription. Still more, the genes-encoded by operon icsB-ipaCB seem to be regulated of a distinct form. Therefore, transcription of the ipaD in EIEC probably depends on distinct signaling way of S. flexneri. New studies shows to be necessary for the better understanding of the pathogenic mechanism of EIEC.

As infecções causadas por patógenos intestinais gram-negativos são importantes fontes de prejuízos na criação de animais domésticos como bovinos, ovinos, suínos e aves. Em muitos casos, opta-se pela administração de antibióticos de amplo espectro para prevenção destas infecções e atenuação das perdas. No entanto, esta prática apresenta risco para a saúde humana, bem como contribui para seleção de cepas resistentes. Nos últimos anos, muito tem se discutido a respeito de novas alternativas para atenuar os prejuízos mencionados sem apresentar o mesmo risco. Uma destas alternativas é a utilização de micro-organismos que apresentam propriedades probióticas que sintetizam substâncias inibitórias sobre espécies de patógenos intestinais. Esta abordagem praticamente elimina o risco de desenvolvimento de resistência aos antibióticos de uso clínico, além de evitar a presença de resíduos nos produtos de origem animal. O termo \"probiótico\" é utilizado atualmente para definir micro-organismos que, administrados em doses e frequências adequadas, conferem benefícios à saúde do hospedeiro. Nos últimos anos, diversos estudos têm sido desenvolvidos envolvendo quatro cepas de Lactobacillus (L. acidophilus ATCC 4356, L. casei ATCC 7469, L. fermentum ATCC 9338 e L. plantarum ATCC 8014) com resultados promissores no tocante às suas características probióticas. Desta forma, a proposta deste estudo foi avaliar a capacidade destas cepas em relação à produção de substâncias que apresentam atividade inibitória sobre espécies patogênicas, intestinais gram-negativas, especificamente Escherichia coli 0112, Escherichia coli 0124, Escherichia coli 0127, Shigella sonnei ATCC 25931, Shigella dysenteriae ATCC 13313, Salmonella enteritidis ATCC 13076. Para tanto, foi avaliada a atividade inibitória de substâncias presentes no sobrenadante livre de células de cada cepa. Adicionalmente, foi realizada a caracterização presuntiva das substâncias responsáveis pela inibição do crescimento microbiano quando os respectivos sobrenadantes foram submetidos a diferentes tratamentos (catalase, enzimas proteolíticas, tratamento térmico e neutralização do pH). A estratégia adotada consistiu na avaliação do crescimento, determinado por turbidimetria, das cepas patogênicas na presença do sobrenadante in natura e tratado. Os valores de absorbância foram analisados estatisticamente pelos testes de ANOVA e Tukey e os resultados mostraram que os sobrenadantes in natura de L. acidophilus ATCC 4356 L. casei ATCC 7469 e L. plantarum ATCC 8014 foram capazes de inibir 5 das 6 cepas dos patógenos intestinais estudadas em níveis de inibição variando de 23% a 53%, sendo que a cepa S. dysenteriae ATCC 13313 não teve seu crescimento inibido pelas cepas de Lactobacillus avaliadas. Demonstrou-se também que apenas o sobrenadante in natura de L. fermentumATCC 9338 apresentou atividade inibitória sobre esta cepa, cujo percentual de inibição variou entre 20% e 35% e entre 36% e 65% para as demais cepas patogênicas. A avaliação dos resultados relativos ao crescimento das cepas patogênicas na presença dos sobrenadantes in natura e tratados revelou que o efeito de inibição observado foi devido a presença de ácidos orgânicos que provocou o abaixamento do pH. Demonstrou-se ainda a ausência de substâncias peptídicas e termolábeis ativas contra as cepas patogênicas avaliadas, bem como peróxido de hidrogênio nos respectivos sobrenadantes.
Infectious diseases caused by gram-negative pathogens are important sources of losses in livestock, especially bovine, ovine and poultry. In many cases, subclinical administration of broad-spectrum antibiotics is chosen as an approach for the prevention of these infections, consequently decreasing these losses. However, this practice presents an important risk to human health, as well as it contributes to the selection of bacterial strains which are resistant to antibiotics usually employed in clinical practice. Therefore, special attention has been given to finding alternative procedures to decrease those losses while eliminating that risk. One of these alternatives consists of using microorganism species which present probiotic properties such as synthesis of inhibitory compounds that act on intestinal pathogen species. This alternative virtually eliminates the risk of developing resistance to broad-spectrum antibiotics, as well as avoids the presence of antibiotic residues in animal products. The term \"probiotic\" is currently used to define microorganism species which promote several benefits to the host, once they are administered frequently and in adequate amounts. In the last few years, several works have been carried out using four Lactobacillus strains (L.acidophilus ATCC 4356, L.casei ATCC 7469, L. fermentum ATCC 9338 and L. plantarum ATCC 8014), and the results have been satisfactory regarding to their probiotic characteristics. Therefore, the aim of this study was to evaluate the ability of these strains to produce inhibitory compounds which are active on gram-negative intestinal pathogen species, specifically Escherichia coli 0112, Escherichia coli 0124, Escherichia coli 0127, Shigella sonnei ATCC 25931, Shigella dysenteriae ATCC 13313 and Salmonella enteritidis ATCC 13076. So, antimicrobial activity of the cell-free supernatant of each strain was evaluated. Additionally, presumptive characterization of these compounds was undertaken by submitting the supernatants to different treatments (catalase, proteolytic enzymes, thermic treatments, pH neutralizing). The strategy consisted of evaluating the growth, estimated by turbidimetry, of the mentioned pathogenic strains in the presence of the original supernatants, as well as in the presence of treated supernatants. Aborbance values were statistically analyzed by means of ANOVA and Tukey\'s test. The results showed that the original supernatants of L. acidophilus ATCC 4356 L. casei ATCC 7469 and L. plantarum ATCC 8014 were capable of inhibiting five of six strains of enteric pathogens at levels varying from 23% to 53%. S. dysenteriae ATCC 13313 was not inhibited by the Lactobacillus strains evaluated. It was also demonstrated that only the original supernatant of L. fermentum ATCC 9338 showed inhibitory activity upon this strain varing from 15% to 32%, and between 36% and 65% regarding to the other strains. Growth evaluation of the pathogenic strains in the presence of the treated and original supernatants revealed that the inhibition effect observed occurred due to the presence of organic acids, which lowered the pH of the supernatants. It was also demonstrated the absence of hydrogen peroxide, peptidic and thermolabile compounds in the supernatants.

Escherichia coli enteroinvasora (EIEC) e Shigella spp. causam disenteria bacilar, caracterizada pela destruição das células epiteliais da mucosa do cólon do hospedeiro. Ambos os microrganismos apresentam características bioquímicas, genéticas e patogênicas semelhantes, porém a doença causada por EIEC se apresenta numa forma mais branda e auto limitante. Dois genes plasmidiais, icsA e icsB estão envolvidos na disseminação intra e intercelular da bactéria, fator importante na produção e resolução da doença. Trabalhos anteriores mostraram que S. flexneri M90T possui uma capacidade maior de disseminação do que o sorotipo de EIEC O124:H-. Devido a esses resultados surgiu a seguinte pergunta: Será que essas diferenças moleculares e fenotípicas estariam restritas ao sorotipo O124:H- ou é comum ao patotipo EIEC? Assim, neste trabalho avaliamos as características fenotípicas e moleculares de onze diferentes sorotipos de EIEC e as comparamos com as amostras de S. flexneri. Pelo ensaio de placas de lise em células Caco-2 mostramos que a capacidade de disseminação de todos os sorotipos de EIEC é menor quando comparada à Shigella M90T. Ao avaliar as sequências gênicas vimos polimorfismos dos genes icsA e icsB dentro do grupo EIEC, assim como em relação aos sorotipos S. flexneri 2a e S. flexneri 5a. A menor disseminação, apresentado pelo patotipo de E. coli, pode estar associada com o processo de ligação e/ou recrutamento de N-WASP, como também na interação com outras proteínas do hospedeiro.
Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella spp share many genetic and biochemical similarities, the illness caused by EIEC is less severe. The effector proteins IcsA and IcsB are important in the physiopathology of the disease triggered by EIEC and Shigella spp. IcsA is required for intracellular actin-based motility, and the role of IcsB is to camouflage IcsA from the autophagic host defense system. Previous studies showed that EIEC O124:H- showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Due to these results the following question arose: Are molecular and phenotypic differences restricted to serotype O124:H- or is it common to EIEC pathotype? Thus, this study evaluated the phenotypic and molecular characteristics of eleven different serotypes of EIEC and compares them to samples of S. flexneri. All EIEC serotypes presented lower cell-to-cell Caco-2 dissemination compared to Shigella M90T, and the differences between this two species expanded to the icsA and icsB gene sequences, in which it was possible to observe a polymorphism of the genes. The smallest spread presented by EIEC E. coli pathotype could be associated with the connection process and/or recruitment of N-WASP, as well as to other important host proteins.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados.
The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.

Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-α, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença.
In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-α, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.

Este estudo teve por objetivo avaliar a ação inibitória de duas soluções de quitosana através da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de duas soluções de quitosana de diferentes procedências. A primeira solução de quitosana derivada de camarão, do tipo comercial Fluka, de PM 600.000 g/mol, G.A. 76% e a segunda solução de quitosana derivada de lula, de PM: '10 POT.7' g/mol e G.A.: 83%. As duas soluções foram ajustadas ao pH 5,0 e na concentração de 0,5% em solução acética a 1%. A melhor atividade antimicrobiana da quitosana ocorre em pH igual ou menor que cinco e ela sofre precipitação em pH superiores a 6,5. Estas duas características foram determinantes na escolha do pH utilizado no teste da CIM. Para confirmar que a inibição do crescimento dos enteropatógenos ocorreu pela ação da quitosana e não pelo pH ácido dos meios, vários testes foram realizados. A avaliação do crescimento dos enteropatógenos em ágar MacConkey, pH 5,0 (ótimo para quitosana) e 7,4(ótimo para o cultivo das bactérias utilizadas), o inóculo de cada bactéria foi preparado segundo o tubo 0,5 de Mc Farland (controle positivo), e a avaliação foi repetida utilizando o inóculo diluído 1:1000 para contagem do número de colônias, e não apresentaram diferenças significativas. Para confirmar estes dados foram realizados os controles negativos das duas soluções de quitosana e do meio de cultura MacConkey em pH 5,0 e 7,4, incubados a 37°C e lidos por 72 horas, sem qualquer alteração. A avaliação da reação de precipitação foi feita em caldo Müeller Hinton em pH 4,0; 5,0; 6,0; 7,0 e 8,0 para as duas soluções de quitosana (v/v), incubados a 37°C e lidos por 72 horas. A avaliação da CIM das duas soluções de quitosana para os enteropatógenos foi realizada por diluição seriada e os inóculos comparados ao tubo 0,5 de Mc Farland, sendo 10 'mü'L da suspensão bacteriana acrescida a cada tubo, incubados a 37°C por 24 horas. A leitura da ausência de turvação visível caracterizou a CIM. Todos os tubos que não apresentaram turvação visível foram plaqueados em ágar MacConkey, em pH 7,4 e incubados a 37°C por 24 horas para determinação da CBM, a qual foi determinada pela menor concentração capaz de causar morte total da população dos enteropatógenos. Todas as técnicas foram realizadas em triplicata para confirmação dos resultados
The aim of this study was to evaluate the inhibitory action of chitosan solutions derived from shrimp (Fluka commercial type, MW 600.000 g/mol, acetylation degree of 76%) and squid (MW '10 POT.7' g/mol, acetylation degree of 83%) through determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against enteropathogens: Salmonella enterica, Shigella sonnei and Escherichia coli EPEC. Those solutions were set to pH 5,0 and a 0,5% concentration in a 1% acetic acid solution. The best antimicrobial activity of chitosan occurs in pH less than or equal to 5,0 and it shows precipitation in pH greater than 6,5. Those features were decisive to choose the pH used in the MIC test. In order to confirm that the growth inhibition of enteropathogens occurred by the action of chitosan and not for the acid pH of the environments, growth evaluation tests of enteropathogens were accomplished in MacConkey agar, pH 5,0 (excellent for chitosan) and pH 7,4 (excellent for culture of used bacteria). The inoculum of each bacterium was prepared comparing with the 0,5 tube of McFarland (positive control) and the evaluation was repeated using the inoculum diluted in a salt solution 1:1000 to count the number of colonies, which did not show significant differences. The reaction evaluation of precipitation of chitosan was done in Müeller Hinton broth with pH ranging 4,0 - 8,0 for both solutions of chitosan (v/v), which were incubated at 37°C and read for 72 hours. The MIC evaluation for both solutions of chitosan for the enteropathogens was done by serial dilution and the inocula were compared to the 0,5 tube of McFarland, adding 10 'mü'L of bacterial suspension to each tube, which were incubated at 37°C for 24 hours. The MIC was distinguished by the absence of visible turbidity. Each tube that did not show visible turbidity was spread on MacConkey agar plates in pH 7,4, and incubated at 37°C for 24 hours to find the MBC, which was determined by the smallest concentration able to cause total death to the enteropathogen population. In both cases, the solutions of chitosan presented a high antimicrobial activity against the enteropathogens Salmonella enterica and Escherichia coli EPEC. However, the higher antimicrobial activity was observed in the enteropathogen Shigella sonnei

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) and Shigella flexneri relies predominantly on members of the AraC/XylS family of transcriptional regulators, Rns (or its homolog, CfaD) and MxiE, respectively. Rns/CfaD regulate the expression of pili, which allow the bacteria to attach to the intestinal epithelium. Better understanding of the role Rns plays in virulence was attained by expanding our knowledge of the Rns regulon, revealing that it functions as an activator of cexE, a previously uncharacterized gene. By in vitro DNase I footprinting two Rns-binding sites were identified upstream of cexEp, both of which are required for full activation of cexE. The amino terminus of CexE also contains a secretory signal peptide that is removed during translocation to the periplasm. Though the function of CexE remains unknown, these studies suggest that CexE is a novel ETEC virulence factor since it is regulated by Rns/CfaD. In Shigella flexneri, the expression of a subset of virulence genes (including, ipaH9.8 and ospE2) is dependent upon the activator MxiE and a cytoplasmic chaperone IpgC. To define the molecular mechanism of transcriptional activation by this chaperone-activator pair, an in vitro pull down assay was performed revealing that MxiE specifically interacts with IpgC in a complex. Additionally, IpgC recognizes three polypeptide regions in MxiE: within MxiE(1-46), MxiE(46-110) and MxiE(196-216). Furthermore, it seems that MxiE and IpgC regulate transcription of ipaH9.8 and ospE2 promoters differently. In the bacterium, the formation of the MxiE-IpgC complex is initially prevented because IpgC is sequestered in individual complexes with effector proteins, IpaB and IpaC. Upon contact with an eukaryotic host cell the effector proteins are secreted, thereby freeing IpgC to form a complex with MxiE and activate the expression of virulence genes. This new characterization of the role of Rns and MxiE in virulence gene regulation in ETEC and S. flexneri, respectively will give new insights into the pathogenesis of the regulators.

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar, caracteriza-se pela destruição do epitélio do cólon provocado pela resposta inflamatória induzida após invasão da mucosa por bactérias. Cepas de EIEC são bioquímica, genética e patogênica semelhante a Shigella spp. A patogenicidade de EIEC e Shigella dependem da presença da pInv plasmídeo, que contém os genes necessários para a colonização bacteriana na mucosa intestinal. Recentemente, demonstrou-se que genes plasmidias ospC1, ospG e ospF de S. flexneri estão envolvidos na inibição da resposta inflamatória em células epiteliais intestinais, um fator importante na iniciação da colonização bacteriana e produção de doença. Como a EIEC mostrou doença menos grave, foi analisada as sequências de aminoácido, avaliada a transcrição destes genes plasmídiais e resposta inflamatória modulada por este micro-organismo na célula epitelial intestinal Caco-2. As células Caco-2 foram infectadas em momentos diferentes com 11 sorotipos de EIEC e S. flexneri (M90T). Os dados sobre a capacidade de invasão e sobrevivência de bactérias, expressão de genes de bactérias e da quimiocina IL-8 foram obtidos por CFU, RT-PCR, e ELISA, respectivamente. A significância estatística foi avaliada por ANOVA de dois fatores. Os 11 sorotipos de EIEC estudados apresentaram similaridade de 100% com S.flexneri para OspC1 e OspF,contudo, foram diferentes na homologia do OspG. Quando comparamos as sequências de aminoácido dos 11 sorotipos observamos 100% de similaridade entre eles para OspG, sugerindo o envolvimento destas proteínas na modulação da resposta imune induzida por estes micro-organismos. Os sorotipos de EIEC apresentam diferenças na capacidade de invasão dos enterócitos. Algumas diferenças significativas foram observadas na transcrição dos genes e na produção de IL-8. Os sorotipos de EIEC O29: H-e O167: H-apresentou um baixo transcrição de genes ospC1 e ospF, e um aumento significativo na produção de IL-8 quando comparado com outros sorotipos. Além disso, demonstrou que a maior transcrição destes genes por alguns sorotipos de EIEC parecem estar relacionados com a menor indução de IL-8. Estes dados sugerem que as proteínas OspC1 e OspF desempenham um papel na resposta inflamatória. No entanto, não se observou relação na transcrição ospG para a produção de IL-8. Estes resultados sugerem que as proteinas efetoras OspC1 e OspF estão envolvidas na inibição da resposta inflamatória em células epiteliais do intestino favorecendo a invasão da EIEC.
Enteroinvasive E. coli (EIEC) is one of the etiological agents of bacillary dysentery, it is characterized by the destruction of the colonic epithelium caused by the inflammatory response induced upon invasion of the mucosa by bacteria. Strains of EIEC are biochemical, genetic and pathogenic similar to Shigella spp. The pathogenecity of EIEC and Shigella depend on the presence of the plasmid pInv, which contains the genes necessary for bacterial colonization in the intestinal mucosa. Recently, it was demonstrated that the plasmid genes ospC1, ospG and ospF of S. flexneri are involved in inhibition of the inflammatory response in intestinal epithelial cells, an important factor in the initiation of bacterial colonization and production of disease. As EIEC has showed less severe disease, we evaluated the transcription of these plasmid genes and inflammatory response modulated by this microorganism in the intestinal epithelial cell Caco-2. The Caco-2 cells were infected in different times with 11 serotypes of EIEC and S. flexneri M90T strain. The data about sequences of amino acids, invasiveness and survival of bacteria, bacterial genes expression, and chemokine IL-8 were obtained by CFU, RT-PCR, and ELISA, respectively. The statistical significance was evaluated by two-way ANOVA. All EIEC serotypes studied showed 100% similarity with S.flexneri to OspC1 and OSPF, however, were different in the homology of OspG. Compared the amino acid sequences of the 11 serotypes observed 100% similarity between them to OspG, suggesting the involvement of them in modulating of the immune response induced by these microorganisms. There were no differences in the invasion the enterocytes among EIEC serotypes. However, some significant differences were observed in the transcription of those genes and production of IL-8. The EIEC serotypes O29:H- and O167:H- showed a low transcription of genes ospC1 and ospF, and a significant increase in production of IL-8 when compared with other serotypes. Furthermore, it was shown that the high transcription of ospF and ospC1 by some EIEC serotypes are related to low induction of IL-8. These data suggested that the proteins OspC1 and OspF play a role in the inflammatory response. However, we did not observed association between ospG transcription to the production of IL-8. These results lead us to believe that the effector proteins OspF and OspC1 are involved in inhibition of the inflammatory response in intestinal epithelial cells favoring the EIEC invasion.

Thesis (M.S.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (Feb. 23, 2007). Includes bibliographical references.

Many Gram-negative bacterial pathogens such as Shigella and adhesive invasive Escherichia coli (AIEC) cause infections characteristic of hyperinflammation. These infections require antimicrobial therapy. However, due to the widespread emergence of multiple drug-resistant strains, alternative strategies must be sought to combating infectious diseases. It has been shown that natural compounds such as propolin D are able to control Shigella growth inside host cells. Geraniol is another natural product which has a chemical structure similar to the side chain of propolin D, which possesses properties potentially useful for antimicrobial therapy. qPCR analysis revealed that propolin D caused extensive bacterial envelopE stress, as indicated by a changed expression of key bacterial genes involved in stress responses. Propolin D also enhanced the autophagy activity of the host cells; the intracellular growth of S. sonnei was significantly reduced in wild type HEK293 cells but not changed in ATG5 knockout cells. Propolin D was unable to enhance septin cage as intracellular S. sonnei formed actin tails in the presence of propolin D; septin cage would restrict formation of actin tails. Geraniol has been shown to target the major virulence regulator, DsbA, which is vital for Shigella’s survival in the reducing host cell cytosol. Geraniol and geranylxvacetate inhibited DsbA function in vitro; wild type DsbA efficiently reduced fluorescently labelled Di-E-GSSG whereas a mutant protein, DsbA33G, was less potent in this in vitro assay. By supplementing acidic and nutrient-poor medium with geraniol the growth of S. sonnei and AIEC strains was severely inhibited. Geraniol was effective in protecting of Galleria. mellonella larva from S. sonnei and AIEC infection. The Galleria mellonella larvae were highly tolerant to geraniol – indicating the great potential of geraniol for future in vivo and clinical studies. In light of previous reports that geraniol synergistically works with antibiotics and induces IL-10 from macrophages, it was concluded that geraniol holds great potential in treating Shigella and AIEC infections.

Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC.
Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.

La maladie d'Alzheimer (MA) est une maladie neurodégénérative associée au vieillissement, constituant un problème de santé publique majeure dans le monde. Il n’y a actuellement aucun traitement efficace ni de moyen reconnu de prévention de cette maladie. La conception de stratégies préventives est donc indispensable pour lutter contre ces maladies. L’alimentation occidentale est particulièrement riche en acides gras ω-6, notamment en acide arachidonique (ARA), converti en nombreux médiateurs de l’inflammation dans le corps. Des travaux antérieurs du laboratoire ont montré qu’un apport alimentaire en ARA sensibilise les souris à la neurotoxicité des oligomères de peptides Aβ, principal agent de la MA. Dans ce travail de thèse, nous avons étudié l’incidence de cet apport alimentaire en ARA sur le microbiote fécal et l’axe microbiote-intestin-cerveau. Nous avons soumis pendant 9 à 12 semaines des cohortes de 15 souris à 3 régimes alimentaires, un régime conventionnel murin (« Sdt-ARA » de 5% de lipides), un régime modérément hyperlipidique de 15% de lipides dont 31,9% d’acide linoléique (« HL-ARA ») et un régime de 15% de lipides dont 25,3% d’acide linoléique et 6,6% d’ARA (« HL+ARA »). L’analyse du microbiote fécal a révélé que le régime « HL-ARA » favorise la prolifération de l’espèce Bifidobacterium pseudolongum dont les activités bénéfiques contre l’inflammation ont été rapportées, et que l’apport d’ARA supprime cette prolifération en favorisant celle du genre Escherichia-Shigella aux effets potentialisateurs de l’inflammation. Les niveaux d’ARNm des cytokines pro-inflammatoires IL-1β et TNFα ont augmenté entre 3 et 11 fois dans le colon des souris nourries avec le régime « HL+ARA ». D’autres médiateurs de l’inflammation colique comme CD40, adiponectine et ICAM-1 ont vu leur expression augmenter par l’enrichissement en ARA tout comme les marqueurs claudine 1 et occludine qui sont des constituants des jonctions serrées. Ces modifications d’expression suggèrent donc des modifications de l’intégrité de la barrière intestinale. L’apport alimentaire d’ARA a également élevé de plus de 10 fois les niveaux d’ARNm du TNFα dans le tissu adipeux mésentérique alors que le retentissement hépatique a été trouvé beaucoup plus modéré. Au niveau cérébral l’apport alimentaire en ARA entraine une augmentation de 1,5-1,8 fois des niveaux d’expression protéique de la GFAP après 9 semaines de régime dans les hippocampes et le cortex alors que ceux du marqueur microglial Iba1 n’a pas été modifié. Nous avons également mis en évidence une diminution des niveaux d’ARNm de l’IL-6 et du TNFα dans le tissu cérébral global.Ces résultats indiquent que l’apport alimentaire en ARA induit une inflammation de bas grade systémique qui pourrait favoriser l’expansion d’un processus de type Alzheimer dans le cerveau si un signal inducteur survient. A ce titre, nous avons également évalué l’impact d’un modèle d’administration per os innovant de D-galactose rapporté comme étant capable d’induire un vieillissement accéléré, une altération des capacités cognitives et l’apparition de plaques amyloïdes dans le cerveau des souris en élevant le stress oxydant. Nous avons également étudié la survie dans le tube digestif de deux souches de Streptococcus thermophilus dont les travaux antérieurs du laboratoire ont montré l’activité anti-inflammatoire in vitro et d’une souche de Lactobacillus plantarum capable de métaboliser les acides gras polyinsaturés ω-6 en acides gras conjugués. Ce travail de thèse a pour perspective d’évaluer la capacité de ces souches probiotiques à lutter contre l’inflammation de bas grade induite par l’apport alimentaire en ARA et le stress oxydant induit par le D-galactose. De nouveaux travaux sont en cours au laboratoire afin d’investiguer plus en détail l’ensemble des processus biologiques en oeuvre dans les différents modèles et de déterminer l’impact des probiotiques sur une administration conjointe de D-galactose et d’un régime riche en ARA
Alzheimer's disease (AD) is a neurodegenerative disease associated with aging, consisting of a major public health problem worldwide. There is currently no effective treatment or established preventions for this disease, highlighting the importance of the design of preventive strategies in the fight against this disease. Western diets are particularly rich in ω-6 fatty acids, especially arachidonic acid (ARA), which are converted into many inflammation mediators in the organism. Previous laboratory results have shown that dietary intake of ARA sensitizes mice to the neurotoxicity of Aβ peptide oligomers, the main agent of AD. In this thesis work, we studied the impact of this dietary intake of ARA on the fecal microbiota and the microbiota-gut-brain axis. For 9 to 12 weeks, three groups of 15 mice have consumed one of these 3 diets : a conventional murine diet (“Sdt-ARA” of 5% lipids), a moderately high-fat diet of 15% lipids including 31.9% of linoleic acid ("HL-ARA"), and a diet of 15% lipids including 25.3% linoleic acid and 6.6% ARA ("HL+ARA").Analysis of the fecal microbiota revealed that the "HL-ARA" diet promotes proliferation of the Bifidobacterium pseudolongum strain, which has been show to possess anti-inflammatory activities. Furthermore, the supply of ARA suppresses this proliferation by favouring Escherichia-Shigella multiplication (with potentiating effects on inflammation). A 3-to-11-fold increase in mRNA levels of the pro-inflammatory cytokines IL-1β and TNFα has been observed in the colon of mice fed with the "HL+ARA" diet. Enrichment of ARA led to an increase of the expression of other mediators of colon inflammation such as CD40, adiponectin, and ICAM-1, as well as the claudin 1 and occludin markers which are components of cellular tight junctions. Therefore, these changes in expression suggest that the integrity of the intestinal barrier has been compromised. Moreover, dietary intake of ARA led to a 10 fold increase in TNFα mRNA levels in mesenteric adipose tissue, while the impact on the liver was found to be much more moderate. At the cerebral level, a 9 weeks food intake of ARA led to a 1.5-1.8-fold increase in the protein level of GFAP in the hippocampus and the cortex, while no changes of the microglial marker Iba1 has not been reported. We also found decreased levels of IL-6 and TNFα mRNA in the global brain tissue.These results indicate that dietary intake of ARA induces low-grade systemic inflammation which may promote the expansion of an Alzheimer's-like process in the brain if an inducing signal occurs. As such, we also evaluated the impact of an innovative oral administration model of D-galactose, which has been described as an aging accelerator compound , leading to impairment of cognitive capacities, and the appearance of amyloid plaques in the mouse brains through elevated oxidative stress. We also studied the survival in the digestive tract of two strains of Streptococcus thermophilus with anti-inflammatory activity in vitro as reported by previous laboratory work, and of a strain of Lactobacillus plantarum capable of metabolizing ω-6 polyunsaturated fatty acids in conjugated fatty acids. This thesis work aims to assess the ability of these probiotic strains to fight against low-grade inflammation induced by dietary intake of ARAs and oxidative stress induced by D-galactose. New work is underway in the laboratory to investigate in more details all the biological processes at work in the different models and to determine the impact of probiotics on a combined administration of D-galactose and an ARA-enriched diet

La sous unité B de la toxine de Shiga (STxB) est un vecteur vaccinal ciblant les cellules dendritiques. Des réponses lymphocytaires T-CD4 et CD8 ainsi que la production d'anticorps ont été observées après vaccination de souris avec des protéines chimériques associant STxB couplée à différents antigènes. STxB ne favorisant pas la maturation des cellules dendritiques, nous avons évalué l'efficacité vaccinale de STxB mélangée à différents adjuvants. Ainsi, STxB couplée à différents antigènes et associée à l'aGalCer, glycolipide activant les cellules NKT, permet d'augmenter la fréquence de LT-CD8 et de réduire de façon drastique les doses d'antigènes. Cette combinaison vaccinale a également permis de rompre une tolérance à des antigènes du soi et de protéger contre le développement d'une infection virale. Par ailleurs, nous avons montré que la voie d'immunisation influait sur le type de réponses immunitaires (humorale muqueuse, cellulaire systémique) observées après l'utilisation de STxB
The Shiga toxin subunit B (STxB) is a vaccinal vector targeting dendritic cells. CD4+ and CD8+ T cells responses as well as antibody production were observed after vaccination of mice with chimeric proteins composed of STxB coupled with different antigens. STxB doesn't favour maturation of dendritic cells, thus we assessed the STxB efficiency as vector in combination with different adjuvants. STxB coupled with different antigens and mixed with aGalCer, a glycolipide activating NKT cells, resulted in an increase CD8+ T cells frequency and it also allowed the dramaticaly reduction of antigen doses. This vaccine also permitted to break tolerance to self-antigens and to protect against the development of viral infection. In addition, we have showed that the route of immunization had an influence on the type of immune response (mucosal humoral response jind cellular^ systemic response) observed after the use of STxB

The ability to acquire sufficient iron from the environment is essential for growth of most bacteria, including Escherichia coli and Shigella flexneri. In E. coli, the enterobactin-mediated iron acquisition system is the major way for the cells to get iron under iron-limiting conditions. Enterobactin is a siderophore that is synthesized and secreted in response to iron limitation to scavenge external ferric iron with high affinity. In this work, I showed that the alkyl hydroperoxide reductase (Ahp) system participates in cellular iron metabolism in both E. coli and S. flexneri. The Ahp system is composed of two proteins, AhpC and AhpF. AhpC detoxifies peroxides by converting peroxides to alcohol and water, and AhpF recycles AhpC. In this work, the data showed that the ahpC mutant synthesized and secreted much less enterobactin than the wild type E. coli and had a growth defect in low iron medium. AhpC influenced the first step of enterobactin biosynthesis by either facilitating the delivery of its substrate chorismate to the enterobactin biosynthesis pathway, or maintaining an optimal concentration of chorismate inside E. coli cells. In E. coli, the data showed that deletion of both ahpF and the glutathione reductase gor affected iron uptake or utilization, but not enterobactin biosynthesis, indicating the role of AhpF and Gor in cellular iron metabolism is different from that of AhpC. In S. flexneri, the Ahp system was also found to be involved in cellular iron metabolism; however, AhpC was not required for major steps of S. flexneri virulence: invasion, intracellular replication or cell-cell spread. Overall, the Ahp system participated in multiple steps of cellular iron metabolism.
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The aromatic beta-glucosides of plant origin, salicin and arbutin, serve as carbon sources for the sustenance of bacteria when ‘preferred’ sugars are absent in the environment. In the family Enterobacteriaceae, there are varied patterns for utilization of these beta-glucosides, wherein, in some members the ability to utilize salicin or arbutin is cryptic while in others it is completely absent. Escherichia coli harbors silent or cryptic genetic systems for the utilization of arbutin and salicin, which are activated by spontaneous mutation(s). Of these systems, the bgl operon of E.coli has been used as a paradigm for silent genes and extensive studies have been carried out to understand its silencing and activating mechanisms. Mutational activation of the wild type bgl operon in E.coli leads to the acquisition of the ability to utilize both arbutin and salicin. Preliminary studies have shown that aromatic beta-glucoside utilization in Shigella sonnei, which is evolutionarily related to E.coli, shows a two-step activation process wherein the wild type strain first becomes Arb+, which subsequently mutates to Sal+. The genetic systems responsible for beta-glucoside utilization, including the bgl operon, are conserved in S.sonnei to a large extent. A major difference is that the bglB gene encoding the phosphor-β-glucosidase B is insertionally inactivated in S.sonnei. As a result, activation of the bgl operon in the first stage leads to expression of the permease, BglF, which along with the phosphor-β-glucosidase A expressed from an unlinked constitutive gene, bglA, confers an Arb+phenotype. Salicin is not a substrate for the enzyme BglA and therefore a second mutational event is required for the acquisition of the Sal+ phenotype. Interestingly, the insertion within bglB is retained in AK102, the Sal+ second step mutant of S.sonnei. Therefore, the locus involved in conferring salicin utilization ability is unknown. However, S.sonnei is not amenable to routine genetic echniques and an E.coli bglB model was generated by creating an insertion in the bglB gene to identify the locus involved in conferring the Sal+ phenotype. Like S.sonnei, this E.coli strain, SD-1.3, also showed a two-step activation process for the utilization of salicin. Utilization of salicin in the Sal+ second step mutant of SD-1.3 could require activation of other silent genetic systems such as the asc operon and the chb operon or mutation in loci such as bglB or bglA. Linkage analysis by P1 transduction showed that activation of the asc operon is required for conferring a Sal+ phenotype in the second step mutant. The asc operon comprises of two genes, ascF encoding a PTS permease and ascB encoding a phosphor-β-glucosidaseB.The Precise mechanism of activation of the asc operon is not known but, it has been speculated that AscG, encoded by an upstream gene, acts as a repressor. Results presented in this thesis show that BglF is responsible for the transport of salicin and AscB provides the phosphor-β-glucosidase B in the Sal+ second step mutant of the E.coli strain SD-1.3. Analysis of the expression of the ascFB operon by measuring the transcripts as well as the activity of phosphor-β-glucosidase B showed that it is enhanced in the Sal+ second step mutant of SD-1.3 in the presence of the inducer. The expression of the ascFB operon is also increased constitutively when ascG is replaced by an antibiotic cassette in the parent strain SD-1.3 and the Arb+ first step mutant, indicating that AscG acts as a repressor for the asc operon. Moreover, inactivation of ascG in the parent leads to utilization of salicin in a single step by the activation of the bgl operon to provide the transport function, indicating that the inactivation of ascG is sufficient to activate the expression of ascB. Similarly, loss of AscG–mediated repression of the asc operon confers salicin utilization ability to the Arb+ first step mutant of SD-1.3. Interestingly, measurement of phosphor-β-glucosidase B activity in a Sal+ second step mutant derivative deleted for ascG showed a constitutive increase in the expression of the ascFB operon. Thus, AscG mediates the induction of the asc operon in response to salicin. In order to study the mechanism of activation of the asc operon, the ascB gene was cloned from the Arb+ first step mutant and the Sal+ second step mutant of SD-1.3 in a low copy number vector. Both these constructs were able to confer a Sal+ phenotype to the Arb+ first step mutant indicating absence of any genetic change in ascB in the Sal+ second step mutant. This was also confirmed by sequencing of ascB gene from the strains that showed no changes in the nucleotide sequence. Absence of any insertions within ascG showed that activation of the ascoperon is not achieved through disruption of ascG in the Sal+ second step mutants analyzed. AscG belongs to the GalR family of repressors in which some members require a mutation to enable the binding of sugar to mediate induction. Nucleotide sequence analysis showed that there was no change in the ascG gene in the Sal+ mutants analyzed. However, when the upstream regulatory region of the ascFB operon was analyzed a mutation was found in the -10 sequence of the putative promoter of the ascFB genes. This change leads to a stronger promoter as it brings the -10 sequence closer to the consensus sequence. Therefore, salicin utilization is achieved in the Sal+ second step mutant analyzed by an increase in expression of the asc operon by a promoter-up mutation. The negative effect of binding of AscG on expression of the ascFB operon is relieved in presence of the inducer, salicin. The possible role of the asc operon in salicin utilization in S.sonnei was tested by replacing the ascB gene by anantibiotic cassette in AK102, the Sal+ second step mutant of S. sonnei. This did not lead to loss of salicin utilization. By gene targeting approach it was also found that none of the phosphor-β-glucosidases known in E.coli are involved in degradation of salicin in AK102. A search of the S. sonnei genome database indicated the presence of two putative phosphor-β-glucosidases encoded by glvG and SSO1595. Replacement of glvG gene by anantibiotic cassette in AK102 did not lead to loss of salicin utilization. However, a similar replacement of SSO1595 in AK102 resulted in a Sal+ phenotype indicating that SSO1595 provides the phosphor-β-glucosidase in the Sal+ second step mutant of S. sonnei. A homolog of this enzyme is not present in E.coliorinany of the other members of the Shigella genus. Transcription alanalysis as well as measurement of phosphor-β-glucosidase B activity showed that expression of SSO1595 is enhanced constitutively in AK102. To study the mechanism of mutational activation for achieving salicin utilization in S. sonnei, SSO1595 was cloned from AK101, theArb+ first step mutant and AK102, the Sal+ second step mutant in a low copy numbe rvector. Both these constructs were able to confer a Sal+ phenotype to AK101 indicating an absence of genetic change in SSO1595 in AK102. This was also confirmed by sequencing of SSO1595 gene from the strains. Analysis of the upstream regulatory region of SSO1595 in AK102 indicated a deletion of around 1.0kbp sequence. This was also confirmed by nucleotide sequencing of the region. By primer extension analysis it was found that a new transcriptional start site is generated upstream to the deletion in the Sal+ second stepmutant of S.sonnei. Acquisition of the Sal+ phenotype in AK102 is therefore the resultof the SSO1595 gene being brought under a new promoter as a result of a DNA rearrangement. Overall, this study suggests that a high degree of similarity at the genomic level between organisms does not always ensure similarity in genetic mechanisms as two distinct pathways are responsible for conferring utilization of salicinin S. sonnei and E.coli.

The aromatic beta-glucosides of plant origin, salicin and arbutin, serve as carbon sources for the sustenance of bacteria when ‘preferred’ sugars are absent in the environment. In the family Enterobacteriaceae, there are varied patterns for utilization of these beta-glucosides, wherein, in some members the ability to utilize salicin or arbutin is cryptic while in others it is completely absent. Escherichia coli harbors silent or cryptic genetic systems for the utilization of arbutin and salicin, which are activated by spontaneous mutation(s). Of these systems, the bgl operon of E.coli has been used as a paradigm for silent genes and extensive studies have been carried out to understand its silencing and activating mechanisms. Mutational activation of the wild type bgl operon in E.coli leads to the acquisition of the ability to utilize both arbutin and salicin. Preliminary studies have shown that aromatic beta-glucoside utilization in Shigella sonnei, which is evolutionarily related to E.coli, shows a two-step activation process wherein the wild type strain first becomes Arb+, which subsequently mutates to Sal+. The genetic systems responsible for beta-glucoside utilization, including the bgl operon, are conserved in S.sonnei to a large extent. A major difference is that the bglB gene encoding the phosphor-β-glucosidase B is insertionally inactivated in S.sonnei. As a result, activation of the bgl operon in the first stage leads to expression of the permease, BglF, which along with the phosphor-β-glucosidase A expressed from an unlinked constitutive gene, bglA, confers an Arb+phenotype. Salicin is not a substrate for the enzyme BglA and therefore a second mutational event is required for the acquisition of the Sal+ phenotype. Interestingly, the insertion within bglB is retained in AK102, the Sal+ second step mutant of S.sonnei. Therefore, the locus involved in conferring salicin utilization ability is unknown. However, S.sonnei is not amenable to routine genetic echniques and an E.coli bglB model was generated by creating an insertion in the bglB gene to identify the locus involved in conferring the Sal+ phenotype. Like S.sonnei, this E.coli strain, SD-1.3, also showed a two-step activation process for the utilization of salicin. Utilization of salicin in the Sal+ second step mutant of SD-1.3 could require activation of other silent genetic systems such as the asc operon and the chb operon or mutation in loci such as bglB or bglA. Linkage analysis by P1 transduction showed that activation of the asc operon is required for conferring a Sal+ phenotype in the second step mutant. The asc operon comprises of two genes, ascF encoding a PTS permease and ascB encoding a phosphor-β-glucosidaseB.The Precise mechanism of activation of the asc operon is not known but, it has been speculated that AscG, encoded by an upstream gene, acts as a repressor. Results presented in this thesis show that BglF is responsible for the transport of salicin and AscB provides the phosphor-β-glucosidase B in the Sal+ second step mutant of the E.coli strain SD-1.3. Analysis of the expression of the ascFB operon by measuring the transcripts as well as the activity of phosphor-β-glucosidase B showed that it is enhanced in the Sal+ second step mutant of SD-1.3 in the presence of the inducer. The expression of the ascFB operon is also increased constitutively when ascG is replaced by an antibiotic cassette in the parent strain SD-1.3 and the Arb+ first step mutant, indicating that AscG acts as a repressor for the asc operon. Moreover, inactivation of ascG in the parent leads to utilization of salicin in a single step by the activation of the bgl operon to provide the transport function, indicating that the inactivation of ascG is sufficient to activate the expression of ascB. Similarly, loss of AscG–mediated repression of the asc operon confers salicin utilization ability to the Arb+ first step mutant of SD-1.3. Interestingly, measurement of phosphor-β-glucosidase B activity in a Sal+ second step mutant derivative deleted for ascG showed a constitutive increase in the expression of the ascFB operon. Thus, AscG mediates the induction of the asc operon in response to salicin. In order to study the mechanism of activation of the asc operon, the ascB gene was cloned from the Arb+ first step mutant and the Sal+ second step mutant of SD-1.3 in a low copy number vector. Both these constructs were able to confer a Sal+ phenotype to the Arb+ first step mutant indicating absence of any genetic change in ascB in the Sal+ second step mutant. This was also confirmed by sequencing of ascB gene from the strains that showed no changes in the nucleotide sequence. Absence of any insertions within ascG showed that activation of the ascoperon is not achieved through disruption of ascG in the Sal+ second step mutants analyzed. AscG belongs to the GalR family of repressors in which some members require a mutation to enable the binding of sugar to mediate induction. Nucleotide sequence analysis showed that there was no change in the ascG gene in the Sal+ mutants analyzed. However, when the upstream regulatory region of the ascFB operon was analyzed a mutation was found in the -10 sequence of the putative promoter of the ascFB genes. This change leads to a stronger promoter as it brings the -10 sequence closer to the consensus sequence. Therefore, salicin utilization is achieved in the Sal+ second step mutant analyzed by an increase in expression of the asc operon by a promoter-up mutation. The negative effect of binding of AscG on expression of the ascFB operon is relieved in presence of the inducer, salicin. The possible role of the asc operon in salicin utilization in S.sonnei was tested by replacing the ascB gene by anantibiotic cassette in AK102, the Sal+ second step mutant of S. sonnei. This did not lead to loss of salicin utilization. By gene targeting approach it was also found that none of the phosphor-β-glucosidases known in E.coli are involved in degradation of salicin in AK102. A search of the S. sonnei genome database indicated the presence of two putative phosphor-β-glucosidases encoded by glvG and SSO1595. Replacement of glvG gene by anantibiotic cassette in AK102 did not lead to loss of salicin utilization. However, a similar replacement of SSO1595 in AK102 resulted in a Sal+ phenotype indicating that SSO1595 provides the phosphor-β-glucosidase in the Sal+ second step mutant of S. sonnei. A homolog of this enzyme is not present in E.coliorinany of the other members of the Shigella genus. Transcription alanalysis as well as measurement of phosphor-β-glucosidase B activity showed that expression of SSO1595 is enhanced constitutively in AK102. To study the mechanism of mutational activation for achieving salicin utilization in S. sonnei, SSO1595 was cloned from AK101, theArb+ first step mutant and AK102, the Sal+ second step mutant in a low copy numbe rvector. Both these constructs were able to confer a Sal+ phenotype to AK101 indicating an absence of genetic change in SSO1595 in AK102. This was also confirmed by sequencing of SSO1595 gene from the strains. Analysis of the upstream regulatory region of SSO1595 in AK102 indicated a deletion of around 1.0kbp sequence. This was also confirmed by nucleotide sequencing of the region. By primer extension analysis it was found that a new transcriptional start site is generated upstream to the deletion in the Sal+ second stepmutant of S.sonnei. Acquisition of the Sal+ phenotype in AK102 is therefore the resultof the SSO1595 gene being brought under a new promoter as a result of a DNA rearrangement. Overall, this study suggests that a high degree of similarity at the genomic level between organisms does not always ensure similarity in genetic mechanisms as two distinct pathways are responsible for conferring utilization of salicinin S. sonnei and E.coli.

碩士
國立中興大學
食品科學系
87
The presence of Escherichia coli has traditionally been considered an indicator microorganism of fecal contamination in food and in environmental water, and few of them are pathogenic to cause serious disease and food poisoning. So E. coli is therefore a widely used measure of sanitary quality. The common method for detecting and quantifying E. coli requires a minimum of 5 to 7 days to obtain results after initiation of sample analysis. Incorporated MUG to culture broth (i.e MUG-LST) were developed, but it could yield false-positive and false-negative results. The time and accuracy limitation suggested the need for more rapid methods for detecting E. coli. The first purpose of the study is to design PCR primers for the specific detection of the mdh gene of E. coli strains. Positively amplified DNA bands generated for all tested E. coli and Shigella spp. cells collected in our laboratory, and non-E. coli strains would not produce the specific PCR product. The detection sensitivity of Emdh1/ Emdh2 primers (25 pmole each) was 100 CFU per assay. In detection of E. coli cells in milk samples, tap water, underground water and lake water, the detection sensitivity would be 100 CFU if a pre-enrichment step using MacConkey broth was performed prior to the PCR. Owing the DNA relationship of E. coli and Shigella are homologous (about 70%~ 100%) in molecular level was reported. So the second work were sequencing Emdh1/ Emdh2 PCR products and structural gene in mdh gene of Shigella spp. for understanding the similarity. The results showed that the homology of E. coli and Shigella spp. was up to 98%. On the third part of the work, we have tried to develop PCR methods for the differentiation of viable and non-viable cells. By short term culture of the target cells followed by suitable dilution and PCR assay, non-viable cells could be differentiated from the viable cells since non-viable cells could not grow and its DNA would not give any PCR product due to dilution out of the cells. The detection sensitivity of viable cells were 100 CFU per assay, but the detection sensitivity of non-viable cells treated with autoclave sterilized, boiled-killing, Presterization and 70% EtOH were 102 CFU per assay. If a 8 hours pre-enrichment step was performed prior to PCR, could not generate target PCR products even the original non-viable cells number were 105 cells. Finally, mdh gene specific PCR primers Emdh1/ Emdh2 and MDH31/ MDH2 were combined into a multiplex PCR system and used for the simultaneous detection of E. coli and Salmonella typhimurium. On detection of E. coli and S. typhimurium cells in whole milk and drinking water, if a 8 hours pre-enrichment step was performed prior to PCR, the detection sensitivity for target cells in samples would be reach to 100/ 100 CFU per assay. When two target strains were different numbers mixed to the food samples, however, it was found that the ratio of original cell numbers should be within 102 so that the target strains with lower cell numbers could be detectable.

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology.
Water shortages, water pollution and climate changes are highly interrelated global issues. These have raised immense concerns about serious adverse effects on the quality, treatment and re-use of wastewater. A major role of water is for vitality of life on earth. Water is recognized as source of evolution from origin to degree of civilization, since it is an essential resource its treatment becomes a necessity for day to day for life. Nanoparticles and their application in treatment of wastewater is becoming a major area of research. It is mainly applicable to the removal of major contaminants like microorganisms. This study was carried out with an objective to investigate the antibacterial and antifungal potentials of nanoparticles. Cobalt and cobalt complexes of urea and thiourea were synthesized and characterized using UV-Vs, PL, FTIR, TEM, SEM, XRD and TGA techniques. The Co particles are in a mixture of rod, agglomerates with irregular shape around 50 – 100 nm in diameter. The Co/Thiourea particles appear to be around 10 – 30nm in size. The Co complexed with urea images showed spherical to hexagonal shape with 50 nm size in diameter. The antimicrobial activity was determined using Minimum Inhibitory and bactericidal concentration and the well diffusion method. The antibacterial and antifungal activities of ratios (1:1, 1:2, 1:3, 2:1 μg/mL) of doped cobalt nanoparticles were tested against a panel of five Gramnegative bacteria - (Escherichia coli, Pseudomonas aeruginosa, Shigella enterica, Salmonella typhi and Salmonella sonnei) human pathogenic bacteria; and two fungal strains - Aspergillus niger and Candida albicans. Zones of inhibition as a consequence of nanoparticles were compared with that of different standards like Neomycin for antibacterial activity and Amphotericin B for antifungal activity. The results showed a remarkable inhibition of the bacterial growth against the tested organisms. The most striking feature of this study is that Cobalt, Urea and Thiourea nanoparticles have antifungal activity comparable or more effective (as in case of Thiourea on A. niger) than Amphotericin B and nearly promising antibacterial activity although not comparable to Neomycin.