Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.     

具核梭杆菌具核亚种蛋白质组及其在癌症pH环境下的差异蛋白质组分析

利用LC-MS/MS对F. nucleatum的蛋白质表达谱进行了深入研究, 共鉴定到1198个蛋白质的表达, 占基因组58%的编码基因. 通过在pH 6.5(癌症组织pH环境)和pH 7.5(正常组织pH环境)两种条件下F. nucleatum蛋白质表达的定量蛋白质组学分析, 发现处于癌症微环境pH条件下蛋白质表达的变化明显集中于金属离子转运, 硫胺素代谢, 糖代谢等过程和功能. 产丁酸发酵的能量代谢通路中的七个代谢酶在蛋白质组数据中找到, 其中有五个在癌症pH条件下显著下调, 这五个酶包含了丁酸盐代谢的整个过程, 这为结直肠癌患者肠道丁酸盐含量降低提供了新的证明. 此外, 癌症pH条件下F. nucleatum的丁酸代谢受到抑制, 造成产丁酸能力下降, 可能是其促进结直肠癌发展在代谢水平上的原因之一.     

A Fluorogenic DNAzyme for A Thermally Stable Protein Biomarker from Fusobacterium nucleatum,a Human Bacterial Pathogen

Fusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter-selection step, we derived an fluorogenic RNA-cleaving DNAzyme, named RFD-FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme-based biosensing directly with biological samples because nucleases found inherently in these samples can be heat-inactivated. We further demonstrate that RFD-FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD-FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.     

The Fruits of Paris polyphylla Inhibit Colorectal Cancer Cell Migration Induced by Fusobacterium nucleatum-Derived Extracellular Vesicles

Colorectal cancer (CRC) is one of the most common cancers worldwide. Gut microbiota are highly associated with CRC, and Fusobacterium nucleatum was found to be enriched in CRC lesions and correlated with CRC carcinogenesis and metastases. Paris polyphylla is a well-known herbal medicine that showed anticancer activity. The present study demonstrates that P. polyphylla inhibited the growth of CRC cells. In addition, treating with active compounds pennogenin 3-O-beta-chacotrioside and polyphyllin VI isolated from P. polyphylla inhibited the growth of F. nucleatum. We also found that extracellular vesicles (EVs) released from F. nucleatum could promote mitochondrial fusion and cell invasion in CRC cells, whereas active components from P. polyphylla could dampen such an impact. The data suggest that P. polyphylla and its active ingredients could be further explored as potential candidates for developing complementary chemotherapy for the treatment of CRC.