Kawamura Ayaka, Nishikawa Kiyoshi, Iida Haruka, Miyazawa Ken, Goto Shigemi, Hasegawa Yoshiaki
Department of Orthodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Japan.
Department of Microbiology, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.
J Oral Biosci. 2023 Mar;65(1):80-87. doi: 10.1016/j.job.2022.11.001. Epub 2022 Nov 12.
Porphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis.
Comparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR).
Five protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase.
The AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.
牙龈卟啉单胞菌是一种关键的牙周病原体，具有多个双组分系统，被认为可调节毒力。在本研究中，我们聚焦于PGN_0775应答调节因子（RR），一种AtoC同源物，并试图鉴定其在牙龈卟啉单胞菌中调控的靶基因。
采用二维电泳和肽质量指纹图谱组成的比较蛋白质组学分析方法，对亲本（野生型）和atoC基因敲除（KO）菌株的总蛋白样品进行筛选，以寻找受影响的蛋白斑点。使用相对定量实时聚合酶链反应（RQPCR）进一步确认相应基因表达的波动情况。
在pgn_0775 KO菌株中鉴定出五个表达水平波动的蛋白斑点，以及它们的质量和生理特征，其中包括两种在野生型中表达水平高于KO菌株的假定蛋白。RQPCR分析证实，KO菌株中的mRNA水平持续下降，而在pgn_0775互补的KO菌株中恢复。这两种假定蛋白似乎是一个操纵子的产物，该操纵子包含四个基因，编码三种假定但可能含IX型分泌系统分选结构域的蛋白以及C25半胱氨酸蛋白酶的N端区域。
牙龈卟啉单胞菌中的AtoC RR同源物上调了编码保留在细胞表面的潜在抗原蛋白的操纵子的表达；因此，它可能是牙龈卟啉单胞菌特异性抗毒力治疗的一个有前景的靶点。
