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Abstract(s)
Aliarcobacter butzleri é uma espécie de bactérias de Gram-negativo, pertencente ao
género Aliarcobacter e à família Arcobacteraceae. Este microrganismo está distribuído
em diversos ambientes e hospedeiros sendo descrito como um agente enteropatogénico
devido à sua associação com doença diarreica. A patogenicidade de A. butzleri tem sido
destacada pela sua capacidade de aderir, invadir e até sobreviver intracelularmente em
linhas celulares epiteliais do intestino, para além de produzir toxinas e induzir a secreção
de citocinas pró-inflamatórias. O flagelo bacteriano é um conhecido fator de virulência,
com um papel importante em diversos agentes enteropatogénicos. A espécie de A.
butzleri possui um único flagelo polar, cujo filamento possui a proteína flagelina A, que
representa a principal unidade do filamento flagelar e que confere motilidade à bactéria.
Embora tenham sido feitos alguns estudos para elucidar a potencial patogenicidade de
A. butzleri, existe ainda a necessidade de compreender melhor o mecanismo que
desencadeia a infeção por esta bactéria. Assim, este trabalho teve como objetivo avaliar
o papel do flagelo na virulência de A. butzleri. Para tal, começou por se analisar o perfil
de motilidade de diversos isolados de A. butzleri, os quais apresentaram elevada
variabilidade. De seguida, de forma a perceber o comportamento de A. butzleri na
presença de um conjunto de fatores ambientais e outros associados à passagem da
bactéria através do trato gastrointestinal, foi avaliada a motilidade bacteriana de duas
estirpes selecionadas. Assim, a motilidade apresentou-se diminuída em condições de
aerobiose, em concentrações menores de nutrientes e à medida que a temperatura
diminuía, apresentando menor valor de motilidade a 15 ºC. Também, na presença de
mucinas e concentrações mais altas de ácidos gordos de cadeia curta, (90 e 130 mM)
associadas ao intestino grosso, a motilidade diminuiu. Posteriormente, foi avaliada a
expressão do gene flaA, que codifica para a flagelina de A. butzleri, na presença de
compostos associados ao intestino, não sendo no geral observadas diferenças
significativas na expressão do gene nas condições testadas em relação ao controlo. No
entanto, na presença de 30 e 90 mM de ácidos gordos de cadeia curta em relação aos
controlos de NaCl, verificou-se um aumento significativo da expressão de flaA em ambos
os casos. De seguida, avaliou-se o papel funcional do flagelo na virulência de A. butzleri,
através da construção de um mutante por mutagénese insercional no gene flaA e através
de transformação natural. Para tal, foi avaliado o efeito desta mutação na motilidade, na
formação de biofilmes, na velocidade bacteriana, na capacidade de aderir e invadir
células Caco-2 e na capacidade de adesão a mucinas. A estirpe mutante de A. butzleri
DQ40A1?flaA exibiu uma diminuição da motilidade, velocidade média, capacidade de
formar biofilmes, de aderir a mucinas e de invadir a linha celular Caco-2, não reduzindo a sua capacidade de adesão celular, quando comparada com a estirpe nativa. Em suma,
tendo em conta os resultados obtidos podemos afirmar que a influência das condições
ambientais e do hospedeiro podem afetar o perfil de motilidade bacteriana e o flagelo
apresenta um papel importante na mediação da patogenicidade de A. butzleri.
Aliarcobacter butzleri is a Gram-negative bacterium, belonging to the genus Aliarcobacter and the family Arcobacteraceae. This microorganism is distributed in diverse environments and hosts and it is described as an enteropathogenic agent due to its association with diarrheal disease. The pathogenicity of A. butzleri has been highlighted by its ability to adhere, invade and even survive intracellularly in intestinal epithelial cell lines, in addition to producing toxins and inducing the secretion of proinflammatory cytokines. The bacterial flagellum is a known virulence factor with an important role in several enteropathogenic agents. A. butzleri has a single polar flagellum, whose filament contain the flagellin A protein, which represents the main unit of the flagellar filament and confers motility to the bacterium. Although some studies have been done to elucidate the potential pathogenicity of A. butzleri, there is still a need to better understand the mechanism that triggers infection by this bacterium. Thus, this work aimed to evaluate the role of the flagellum in the virulence of A. butzleri. To this end, the motility profile of several isolates of A. butzleri was first analyzed, which showed high variability. Next, in order to understand the behavior of A. butzleri in the presence of a set of environmental factors and others associated with the passage of the bacterium through the gastrointestinal tract, the bacterial motility of two selected strains was evaluated. The results show that motility under aerobic conditions, in lower nutrient concentrations and as the temperature decreased, with the lowest motility value being at 15 ºC. In addition, motility in the presence of mucins and higher concentrations of short chain fatty acids, (90 and 130 mM) associated with the large intestine also diminished. Subsequently, the expression of the flaA gene encoding for A. butzleri flagellin in the presence of gut-associated compounds was evaluated, and overall, no significant differences in gene expression under the tested conditions were observed compared to the control. However, in the presence of 30 and 90 mM of short chain fatty acids a significant increase in flaA expression when compared to NaCl controls. Next, the functional role of the flagellum in the virulence of A. butzleri was evaluated by constructing a mutant by insertional mutagenesis in the flaA gene and by natural transformation. To this end, the effect of this mutation on motility, biofilm formation, bacterial speed, ability to adhere to and to invade Caco-2 cells, and the ability to adhere to mucins were evaluated. The mutant strain of A. butzleri DQ40A1?flaA exhibited decreased motility and mean velocity, less ability to form biofilms, less ability to adhere to mucins as well as to invade Caco-2 cell line, while not reducing its cell adhesion ability, when compared to the native strain. In summary, considering the obtained results, we can state that the influence of environmental and host conditions can affect the bacterial motility profile and the bacterial flagellum plays an important role in mediating the pathogenicity of A. butzleri.
Aliarcobacter butzleri is a Gram-negative bacterium, belonging to the genus Aliarcobacter and the family Arcobacteraceae. This microorganism is distributed in diverse environments and hosts and it is described as an enteropathogenic agent due to its association with diarrheal disease. The pathogenicity of A. butzleri has been highlighted by its ability to adhere, invade and even survive intracellularly in intestinal epithelial cell lines, in addition to producing toxins and inducing the secretion of proinflammatory cytokines. The bacterial flagellum is a known virulence factor with an important role in several enteropathogenic agents. A. butzleri has a single polar flagellum, whose filament contain the flagellin A protein, which represents the main unit of the flagellar filament and confers motility to the bacterium. Although some studies have been done to elucidate the potential pathogenicity of A. butzleri, there is still a need to better understand the mechanism that triggers infection by this bacterium. Thus, this work aimed to evaluate the role of the flagellum in the virulence of A. butzleri. To this end, the motility profile of several isolates of A. butzleri was first analyzed, which showed high variability. Next, in order to understand the behavior of A. butzleri in the presence of a set of environmental factors and others associated with the passage of the bacterium through the gastrointestinal tract, the bacterial motility of two selected strains was evaluated. The results show that motility under aerobic conditions, in lower nutrient concentrations and as the temperature decreased, with the lowest motility value being at 15 ºC. In addition, motility in the presence of mucins and higher concentrations of short chain fatty acids, (90 and 130 mM) associated with the large intestine also diminished. Subsequently, the expression of the flaA gene encoding for A. butzleri flagellin in the presence of gut-associated compounds was evaluated, and overall, no significant differences in gene expression under the tested conditions were observed compared to the control. However, in the presence of 30 and 90 mM of short chain fatty acids a significant increase in flaA expression when compared to NaCl controls. Next, the functional role of the flagellum in the virulence of A. butzleri was evaluated by constructing a mutant by insertional mutagenesis in the flaA gene and by natural transformation. To this end, the effect of this mutation on motility, biofilm formation, bacterial speed, ability to adhere to and to invade Caco-2 cells, and the ability to adhere to mucins were evaluated. The mutant strain of A. butzleri DQ40A1?flaA exhibited decreased motility and mean velocity, less ability to form biofilms, less ability to adhere to mucins as well as to invade Caco-2 cell line, while not reducing its cell adhesion ability, when compared to the native strain. In summary, considering the obtained results, we can state that the influence of environmental and host conditions can affect the bacterial motility profile and the bacterial flagellum plays an important role in mediating the pathogenicity of A. butzleri.
Description
Keywords
Condições Ambientais Aliarcobacter Butzleri Flagelina a Flagelo Hospedeiro Motilidade Virulência