Bhattacharyya Sanghamitra, Ghosh Santosh K, Shokeen Bhumika, Eapan Betty, Lux Renate, Kiselar Janna, Nithianantham Stanley, Young Andrew, Pandiyan Pushpa, McCormick Thomas S, Weinberg Aaron
Department of Biological Science, Case School of Dental Medicine, Cleveland, Ohio, USA.
School of Dentistry, University of California-Los Angeles, Los Angeles, California, USA.
Infect Immun. 2016 Apr 22;84(5):1446-1456. doi: 10.1128/IAI.01311-15. Print 2016 May.
We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.
我们之前从具核梭杆菌（一种口腔革兰氏阴性菌）中鉴定出一种细胞壁相关蛋白，该蛋白可在原代人口腔上皮细胞（HOECs）中诱导人β-防御素2（hBD-2），并将其命名为FAD-I（具核梭杆菌相关防御素诱导剂）。在此，我们报告具核梭杆菌不同菌株对hBD-2的诱导存在差异；ATCC 25586和ATCC 23726诱导产生的hBD-2 mRNA显著多于ATCC 10953。将来自高hBD-2诱导菌株的质粒携带的fadI在ATCC 10953的ΔfadI突变体中进行异源表达，导致hBD-2的诱导水平与高诱导菌株相当，这表明FAD-I是具核梭杆菌在HOECs中诱导hBD-2的主要因子。此外，抗FAD-I抗体可阻断具核梭杆菌对hBD-2的诱导达80%以上。在大肠杆菌中表达的重组FAD-I（rFAD-I）在HOECs中引发的hBD-2转录水平和肽释放水平与从具核梭杆菌ATCC 25586中分离的天然FAD-I（nFAD-I）相似。串联质谱分析显示，nFAD-I和rFAD-I在第16位半胱氨酸残基处均有二酰基甘油修饰。半胱氨酸突变为丙氨酸消除了FAD-I诱导hBD-2的能力。最后，FAD-I对hBD-2表达的激活是通过Toll样受体-1/2（TLR-1/2）和TLR-2/6异二聚体介导的。像FAD-I这样的微生物分子可能会以新的治疗方式被用于增强黏膜表面的宿主固有免疫反应。
