Sasaki Yuko, Matsuo Takehiro, Watanabe Yoshihiro, Iwatsuki Masato, Inahashi Yuki, Nishida Satoshi, Naito Mariko, Shoji Mikio
Department of Microbiology and Oral Infection, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki, Nagasaki, 852-8588, Japan.
Ōmura Satoshi Memorial Institute, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
J Antibiot (Tokyo). 2025 Jan;78(2):90-105. doi: 10.1038/s41429-024-00790-8. Epub 2024 Nov 22.
Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is a key pathogen in chronic periodontitis. P. gingivalis has a type IX secretion system (T9SS) that secretes highly hydrolytic proteinases called gingipains for obtaining peptides as an energy source. Although most T9SS-related proteins have been identified, no specific inhibitor of T9SS has been reported. To screen T9SS inhibitors, we focused on and characterized a minimal liquid medium called mC medium that contains milk casein as the sole protein source. We found that P. gingivalis wild-type strain ATCC 33277 caused cloudiness of mC medium without growth. In mC medium, an alkylating agent, iodoacetamide (IAM) that is an inhibitor of gingipains, and a protonophore, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) that dissipates the proton motive force required for T9SS-mediated secretion, clearly inhibited the increase in turbidity. Moreover, neither the gingipain-null mutant nor the T9SS-deficient mutant caused mC medium cloudiness, suggesting that mC medium cloudiness is dependent on gingipain activity and T9SS. These results indicated that mC medium can be used to assess P. gingivalis gingipain activity and its functional T9SS. Using an assay system with mC medium, we discovered that OM-173αA and OM-173βA in the Ōmura Natural Compound Library and nanaomycin A were probable T9SS inhibitors. The compounds need to be further investigated as tools for analyzing T9SS and as potential therapeutic agents for periodontal disease.
牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，是慢性牙周炎的关键病原体。牙龈卟啉单胞菌具有IX型分泌系统（T9SS），该系统分泌称为牙龈蛋白酶的高度水解蛋白酶，以获取肽作为能量来源。尽管大多数与T9SS相关的蛋白质已被鉴定，但尚未报道T9SS的特异性抑制剂。为了筛选T9SS抑制剂，我们重点研究并表征了一种称为mC培养基的基本液体培养基，该培养基含有酪蛋白作为唯一蛋白质来源。我们发现牙龈卟啉单胞菌野生型菌株ATCC 33277导致mC培养基变浑浊但无生长。在mC培养基中，作为牙龈蛋白酶抑制剂的烷基化剂碘乙酰胺（IAM）和消散T9SS介导的分泌所需质子动力的质子载体羰基氰化物3-氯苯腙（CCCP）明显抑制了浊度的增加。此外，牙龈蛋白酶缺失突变体和T9SS缺陷突变体均未导致mC培养基浑浊，这表明mC培养基浑浊取决于牙龈蛋白酶活性和T9SS。这些结果表明，mC培养基可用于评估牙龈卟啉单胞菌牙龈蛋白酶活性及其功能性T9SS。使用含有mC培养基的检测系统，我们发现大村天然化合物库中的OM-173αA和OM-173βA以及纳那霉素A可能是T9SS抑制剂。这些化合物需要作为分析T9SS的工具和作为牙周疾病的潜在治疗剂进行进一步研究。
