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Characterization of clostridioides difficile biofilm matrix proteins
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Resumo(s)
Clostridioides difficile é um importante agente patogénico, atualmente
reconhecido como uma das principais causas de infeção nosocomial associada ao uso de
antibióticos. No entanto, o aumento das infeções na comunidade e a possibilidade de
transmissão zoonótica têm sido uma crescente fonte de preocupação. Cerca de 25% das
infeções causadas por C. difficile originam episódios de recorrência. C. difficile é
estritamente anaeróbico e capaz de diferenciar esporos. C. difficile forma ainda biofilmes,
onde são também formados esporos. Tanto os biofilmes como os esporos conferem
resistência aos antibióticos e ao sistema imunitário do hospedeiro. Esta combinação torna
difícil a eliminação da bactéria. A matriz extracelular é uma componente crucial do
biofilme, sendo geralmente composta por exopolissacarídeos, DNA extracelular e
proteínas. A composição da matriz do biofilme em C. difficile, bem como os mecanismos
subjacentes à sua formação ainda não são totalmente conhecidos.
Com o objetivo de caracterizar as proteínas da matriz do biofilme, foi identificada
uma estirpe clínica que apresenta uma forte produção de biofilme, 1800 (RT126). Em
comparação com a estirpe de referência, a estirpe 1800 apresentou uma maior taxa de
crescimento e eficiência de esporulação, mas a mesma eficiência de germinação. Os
esporos da estirpe 1800 apresentam um exósporo espesso, irregular e do qual emanam
projecções de aparência fibrilar, em contraste com o manto e exósporo mais fino e
uniforme da estirpe de referência. As proteínas da matriz do biofilme foram extraídas e a
sua identificação por espectrometria de massa revelou serem maioritariamente enzimas
citoplasmáticas. O presente trabalho focou-se no papel da desidrogenase do glutamato
(GDH) e da enolase, duas proteínas moonlight. Foi anteriormente demonstrado que a
presença da GDH extracelular é importante para a colonização por C. difficile e para a
progressão da doença. Com o objetivo de compreender o papel da GDH no biofilme de C.
difficile, procedeu-se à eliminação do gene gluD, codificante para a GDH. A mutação
afetou drasticamente a produção de biofilme e a análise por microscopia de varrimento
revelou um forte decréscimo na densidade da matriz, e na quantidade e dimensões das
fibras que normalmente ligam as células.
Demonstrou-se que a GDH purificada é maioritariamente hexamérica, sendo
ainda visíveis formas de ordem superior, todas cataliticamente ativas.
Surpreendentemente, as mesmas formas de GDH acumulam na matriz, mas são
largamente desprovidas de atividade. A complementação extracelular com a forma
selvagem ou a forma cataliticamente inativa da GDH restaurou a capacidade de formação
de biofilme ao mutante. Com o objetivo de entender o papel da enolase no biofilme, e
uma vez que o gene codificante, eno, é essencial, foi construído um mutante condicional
usando o sistema CRISPRi. Este é, tanto quanto sabemos, o primeiro mutante para a
enolase descrito em C. difficile.
Globalmente, descobriu-se que a GDH possui um papel central na formação do
biofilme de C. difficile e sugere-se que em vez de possuir um conjunto dedicado de
proteínas da matriz, este organismo reutiliza proteínas citoplasmáticas que desempenham
funções moonlight na matriz extracelular do biofilme.
Clostridioides difficile is now recognized as a leading cause of nosocomial infections linked to antibiotic therapy. While historically connected to health-care settings, the rise in community-acquired C. difficile infections (CDI) and the possibility of zoonotic transmission are an increasing concern. About 25% of CDI infections lead to disease recurrence. This organism perseveres in the intestine as biofilms, spores or biofilm-associated spores. The convergence of antibiotic- and immune system resistant biofilms and spores poses a serious threat. The extracellular matrix, a central part of the biofilm, is generally composed of exopolysaccharides, extracellular DNA and proteins. The matrix composition of C. difficile biofilms as well as the mechanisms underlying its formation, are still poorly understood. We selected a strong-biofilm producer, strain 1800 (of ribotype 126), to characterize the biofilm-matrix proteins. In comparison to the reference laboratory strain 630Δerm, strain 1800 presented a higher growth rate and sporulation efficiency, while germination efficiencies were similar in both strains. Spores from strain 1800 present a thicker and irregular exosporium with hair-like projections, contrasting with the thinner coat and exosporium in the reference strain. The biofilm-matrix proteins were extracted and identified by mass spectrometry. The majority of these proteins were found to be cytoplasmic enzymes Here, we focused on the role of NAD-specific glutamate dehydrogenase (GDH) and enolase, two known moonlight proteins. Extracellular GDH was previously shown to have a role in promoting C. difficile colonization and disease progression. In order to understand the role of GDH in C. difficile biofilms, we constructed an in-frame deletion mutant of gluD, coding for GDH. Deletion of gluD drastically impaired biofilm formation and scanning electron microscopy analysis revealed a strong decrease in the matrix density and in the dimension and quantity of fibers interconnecting cells. We demonstrated that purified GDH forms an hexamer but also assembles into higher order species, all of which are enzymatically active. Strikingly, the same forms of GDH accumulate in the biofilm-matrix but are mostly inactive. Extracellular complementation with the wild-type or a catalytic inactive form of GDH restored the wild-type biofilm ability to the gluD mutant. To begin studying the role of enolase in biofilms and since its encoding gene, eno, is essential, we constructed a conditional mutant using CRISPRi silencing. This is, to our knowledge, the first eno mutant described for C. difficile. Overall, we discovered an important role for GDH in C. difficile biofilms and we suggest that instead of producing a set proteins with that specific purpose, this organism uses cytoplasmic moonlight proteins to form the extracellular matrix of biofilms.
Clostridioides difficile is now recognized as a leading cause of nosocomial infections linked to antibiotic therapy. While historically connected to health-care settings, the rise in community-acquired C. difficile infections (CDI) and the possibility of zoonotic transmission are an increasing concern. About 25% of CDI infections lead to disease recurrence. This organism perseveres in the intestine as biofilms, spores or biofilm-associated spores. The convergence of antibiotic- and immune system resistant biofilms and spores poses a serious threat. The extracellular matrix, a central part of the biofilm, is generally composed of exopolysaccharides, extracellular DNA and proteins. The matrix composition of C. difficile biofilms as well as the mechanisms underlying its formation, are still poorly understood. We selected a strong-biofilm producer, strain 1800 (of ribotype 126), to characterize the biofilm-matrix proteins. In comparison to the reference laboratory strain 630Δerm, strain 1800 presented a higher growth rate and sporulation efficiency, while germination efficiencies were similar in both strains. Spores from strain 1800 present a thicker and irregular exosporium with hair-like projections, contrasting with the thinner coat and exosporium in the reference strain. The biofilm-matrix proteins were extracted and identified by mass spectrometry. The majority of these proteins were found to be cytoplasmic enzymes Here, we focused on the role of NAD-specific glutamate dehydrogenase (GDH) and enolase, two known moonlight proteins. Extracellular GDH was previously shown to have a role in promoting C. difficile colonization and disease progression. In order to understand the role of GDH in C. difficile biofilms, we constructed an in-frame deletion mutant of gluD, coding for GDH. Deletion of gluD drastically impaired biofilm formation and scanning electron microscopy analysis revealed a strong decrease in the matrix density and in the dimension and quantity of fibers interconnecting cells. We demonstrated that purified GDH forms an hexamer but also assembles into higher order species, all of which are enzymatically active. Strikingly, the same forms of GDH accumulate in the biofilm-matrix but are mostly inactive. Extracellular complementation with the wild-type or a catalytic inactive form of GDH restored the wild-type biofilm ability to the gluD mutant. To begin studying the role of enolase in biofilms and since its encoding gene, eno, is essential, we constructed a conditional mutant using CRISPRi silencing. This is, to our knowledge, the first eno mutant described for C. difficile. Overall, we discovered an important role for GDH in C. difficile biofilms and we suggest that instead of producing a set proteins with that specific purpose, this organism uses cytoplasmic moonlight proteins to form the extracellular matrix of biofilms.
Descrição
Palavras-chave
Microbiologia médica Biologia molecular Clostridioides difficile Biofilme Proteínas moonlight Desidrogenase do glutamato Enolase