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O intestino humano alberga uma diversa comunidade de bactérias comensais que se sustentam da degradação de polissacáridos dietários, impossíveis de digerir para o ser humano, mas que também conseguem sobreviver, utilizando glicanos produzidos pelo seu hospedeiro. Este ecossistema microbiano, designado de microbiota intestinal, inclui membros da subespécie Bacteroides, nomeadamente Bacteroides caccae e Bacteroides thetaiotaomicron, que possuem Polysaccharide Utilization Loci (PULs) que codificam uma variedade de enzimas ativas para hidratos de carbono (CAZymes). Estas CAZymes são tipicamente modulares e funcionam em conjunção com módulos auxiliares não-cataliticos (CBMs), que reconhecem estruturas de hidratos de carbono específicas e dirigem o domínio catalítico das CAZYmes para estes substratos. Apesar da sua importância, muitos CBMs permanecem estrutural e funcionalmente desconhecidos, limitando o nosso conhecimento acerca das suas interações moleculares.
O objetivo principal do trabalho aqui reportado foi a caracterização de 2 CBMs da família 32 de Bacterodes caccae (BC07580) e Bacteroides thetaioaomicron (BT0865), dois membros prevalentes do microbiota intestinal humano. Com este objetivo em mente, ambos os CBMs foram expressos por métodos recombinantes em Escherichia coli, purificados e preparados para ensaios de cristalização com sucesso. Ensaios de Dicroísmo Circular e Thermal Shift Assays definiram a estrutura secundária e condções tampão mais estáveis de cada CBM, e ensaios de cristalização foram realizados para os dois CBMs na sua forma apo, como incubados com os seus glicanos de maior afinidade.
Devido ao insucesso na obtenção de cristais viáveis, a caracterização do módulo BC07580-C (PUL 36) foi limitada à simulação realizada utilizando o AlphaFold3, enquanto as estruturas 3D de BT0865-C (PUL 12) em complexo com LacDiNAc e LacNAc foram obtidas por cristalografia de raios-X com uma resolução de 1.7Å em ambos os casos.
Estes estudos revelam como estes CBMs operam e fornecem informação valiosa acerca das suas interações no ambiente intestinal humano, e em como estas bactérias comensais interagem com o seu hospedeiro.
The human gut harbors a diverse community of commensal bacteria that contribute to the breakdown of dietary polysaccharides indigestible by humans but also can adapt to thrive using host-derived glycans. This microbial ecosystem, referred to as the gut microbiota, includes members of the Bacteroides spp, such as Bacteroides caccae and Bacteroides thetaiotaomicron, that possess polysaccharide localization loci (PUL) that code for a wide range of carbohydrate-active enzymes (CAZymes). These CAZymes are often modular and function in conjunction with non-catalytic ancillary carbohydrate-binding modules (CBMs), which recognize specific carbohydrate structures and direct the catalytic domains of CAZymes to their substrates. Despite their importance, many CBMs remain structurally and functionally uncharacterized, limiting our understanding of their molecular interactions. The main goal of the work here reported was to structurally characterize two family 32 CBMs from Bacterodes caccae (BC07580) and Bacteroides thetaioaomicron (BT0865), two prevalent members of the human gut microbiota. With this goal in mind, both CBMs were recombinantly expressed in Escherichia coli, purified and prepared for crystallization assays successfully. Circular Dichroism and Thermal Shift assays defined the secondary structure and preferred buffers of each CBM, and crystallization trials of both CBMs unbound and incubated with their ideal glycan partner were performed. The structure of BC07580-C (PUL 36) could only be assessed through simulations performed with AlphaFold3, whereas high-resolution 3D structures of BT0865-C (PUL 12) in complex with LacDiNAc and LacNAc were experimentally determined at 1.7Å resolution using X-ray crystallography methods in both cases. These studies shine a light on how these CBMs operate and provide valuable information about their interactions in the human intestine environment, and how these commensal bacteria interact with their host.
The human gut harbors a diverse community of commensal bacteria that contribute to the breakdown of dietary polysaccharides indigestible by humans but also can adapt to thrive using host-derived glycans. This microbial ecosystem, referred to as the gut microbiota, includes members of the Bacteroides spp, such as Bacteroides caccae and Bacteroides thetaiotaomicron, that possess polysaccharide localization loci (PUL) that code for a wide range of carbohydrate-active enzymes (CAZymes). These CAZymes are often modular and function in conjunction with non-catalytic ancillary carbohydrate-binding modules (CBMs), which recognize specific carbohydrate structures and direct the catalytic domains of CAZymes to their substrates. Despite their importance, many CBMs remain structurally and functionally uncharacterized, limiting our understanding of their molecular interactions. The main goal of the work here reported was to structurally characterize two family 32 CBMs from Bacterodes caccae (BC07580) and Bacteroides thetaioaomicron (BT0865), two prevalent members of the human gut microbiota. With this goal in mind, both CBMs were recombinantly expressed in Escherichia coli, purified and prepared for crystallization assays successfully. Circular Dichroism and Thermal Shift assays defined the secondary structure and preferred buffers of each CBM, and crystallization trials of both CBMs unbound and incubated with their ideal glycan partner were performed. The structure of BC07580-C (PUL 36) could only be assessed through simulations performed with AlphaFold3, whereas high-resolution 3D structures of BT0865-C (PUL 12) in complex with LacDiNAc and LacNAc were experimentally determined at 1.7Å resolution using X-ray crystallography methods in both cases. These studies shine a light on how these CBMs operate and provide valuable information about their interactions in the human intestine environment, and how these commensal bacteria interact with their host.
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Palavras-chave
Gut microbiota Commensal bacteria Carbohydrate-Binding Modules Glycan microarrays X-ray crystallography