Table_2_Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture.PDF

Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are over-expressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1. dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1. Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread.

艰难梭菌（Clostridium difficile）作为一种机会性肠道病原体，能在菌群失调后引发抗生素后和医院相关性腹泻。尽管生物膜可能促进定植，但其发育与生理学尚知之甚少。菌株630Δerm能在连续流动微发酵器中形成大型菌落和附着于玻璃的浸没式生物膜。根据基因表达数据，在生物膜/浮游细胞中，中心代谢活跃并驱动脂肪酸生物合成而非发酵。因此，琥珀酸被消耗，丁酸的产生减少。与代谢协调的毒素A表达被下调，而表面蛋白，如粘附素和主要IV型菌毛亚基，则被过表达。C-di-GMP水平可能通过编码二鸟苷酸环化酶（如dccA）和磷酸二酯酶（如pdstA）的基因表达得到严格调控。c-di-GMP调控的基因表达，编码潜在的表面粘附素CD2831和主要IV型菌毛PilA1，表明c-di-GMP在生物膜细胞中可能较高。一种类似于枯草杆菌（Bacillus subtilis）SinR的调节因子CD2214，以及与CD2214在同一操纵子中共同编码的另一调节因子CD2215，控制着生物膜中差异表达的许多基因，特别是以正调控方式影响dccA、CD2831和pilA1。在微量滴定板培养和破坏后，生物膜由坚固的聚集结构组成，其中细胞嵌入到多形态材料中。原位观察到的完整生物膜呈现出稀疏、异质且高度三维的架构，由杆状结构和微聚集构成。在CD2214和CD2215基因的突变体中，生物膜密度更高，但CD2831和pilA1的失活并未影响生物膜。当dccA过表达时，不仅会增加生物膜，还会使生物膜的架构变得均匀和高度聚集，这种变化独立于CD2831，对pilA1的依赖性极小。细胞微聚集在生物膜形成和架构中发挥着主要作用。这一对基因表达重编程和生物膜架构重塑的详尽分析，为更深入理解这一生活方式奠定了基础，并可能引起新的策略来限制艰难梭菌的传播。