Table 13_The Porphyromonas gingivalis RNA-binding protein is required for growth in high levels of zinc and persistence with host cells.xlsx

The oral periodontal pathogen Porphyromonas gingivalis must adapt to an ever-changing environment to survive and cause disease. So far, most of the efforts concerning the regulatory mechanisms employed by the bacterium centered on DNA-binding regulators. Although global regulatory mechanisms employing RNA-binding proteins (RBP) are reported in most forms of life so far, such mechanism of regulation remains unknown in the oral Bacteroidetes group. Examination of the genome of P. gingivalis led to the discovery of a putative RBP with the RNA recognition motif 1 (RRM-1) designated here RbpPg1 (RNA-binding protein Porphyromonas gingivalis 1). The recombinant form of the protein-bound RNA and RNA-pull down identified a zinc exporter transcript as the most enriched one in agreement with the higher levels of zinc in the absence of the protein. Deletion of RbpPg1 reduced the ability of the bacterium to grow with 0.5 mM zinc. The RgpB protein level and the Arg-X protease activity was reduced in both iron replete and iron deplete conditions in the mutant strain when compared to the wild type. Lys-X protease activity was reduced, although Kgp protein levels were not altered by deletion of RbpPg1. The mutant grew better in hemin-deplete conditions when compared to the wild type. Finally, RbpPg1 was indispensable for the bacterium to survive with host cells. We have determined that both the transcriptome and proteome are affected by the deletion of RbpPg1 and found that the major group of proteins with elevated expression were the ones associated with response to environmental stress changes, while proteins mediating metabolic processes were downregulated. Overall, the first RBP characterized in P. gingivalis plays a significant role in the biology of the bacterium and differs from RBPs in other Gram-negative bacteria. Data are available via ProteomeXchange with identifier PXD034144 and via the NCBI Gene Expression Omnibus (GEO) and under accession number GSE168570.

口腔牙周病原体牙龈卟啉单胞菌（Porphyromonas gingivalis）必须适应不断变化的环境才能存活并致病。迄今为止，有关该细菌调控机制的研究大多聚焦于DNA结合调控因子。尽管目前绝大多数生命形式中均已报道过RNA结合蛋白（RNA-binding protein, RBP）介导的全局调控机制，但这类调控模式在口腔拟杆菌门类群中仍属未知。对牙龈卟啉单胞菌的基因组进行分析后，我们发现了一种带有RNA识别基序1（RNA recognition motif 1, RRM-1）的推定RNA结合蛋白，本文将其命名为RbpPg1（牙龈卟啉单胞菌RNA结合蛋白1）。该蛋白的重组形式可结合RNA，通过RNA下拉实验（RNA-pull down）鉴定得到的富集程度最高的转录本为锌输出蛋白转录本，这与缺失该蛋白时锌水平升高的结果一致。敲除RbpPg1会降低该细菌在0.5 mM锌浓度下的生长能力。与野生型菌株相比，突变株在铁充足和铁匮乏两种条件下的RgpB蛋白水平以及Arg-X蛋白酶活性均有所降低。尽管Kgp蛋白水平未受RbpPg1敲除的影响，但Lys-X蛋白酶活性有所下降。突变株在血红素匮乏条件下的生长情况优于野生型菌株。最后，RbpPg1对于该细菌在宿主细胞内存活必不可少。我们已证实，RbpPg1的敲除会影响细菌的转录组和蛋白质组，且发现表达上调的主要蛋白类群与环境应激响应相关，而参与代谢过程的蛋白则表达下调。总而言之，牙龈卟啉单胞菌中首个被鉴定的RNA结合蛋白在该细菌的生命活动中发挥重要作用，且与其他革兰氏阴性菌中的RNA结合蛋白存在差异。相关数据集可通过ProteomeXchange数据库（标识符PXD034144）以及NCBI基因表达综合数据库（Gene Expression Omnibus, GEO，登录号GSE168570）获取。