Characterization of the effect of a newly identified gene encoding the lipid A deacylase on Porphyromonas gingivalis virulence

摘要 牙龈下革兰氏阴性厌氧菌与牙周炎密切相关 牙周炎，一种慢性炎症性疾病。它在牙周病患者中含量更高。 而不是在健康的地方，这表明它有很好的装备来在敌对的炎症条件下生存下来。此外, 小鼠的感染模型表明，感染牙龈假单胞菌可以使牙龈下倾斜。 微生物群转变为致病的非生物状态。 脂蛋白A是脂多糖的疏水锚，是宿主天然的细菌配体 免疫受体TLR4。在牙龈假单胞菌中，通过脱酰化修饰脂类A，从最初合成的五- 酰化结构到四酰化结构，这是避免TLR4介导的前体结构的关键步骤。 炎症反应。已知的四酰化脂A对细菌的毒力也有很大的贡献 性质，如外膜囊泡(OMV)的形成，以及减轻炎性小体的激活。 牙龈假单胞菌中的OMV与外膜(OM)的不同之处在于它们含有更多的底物-- 脂酰化A，更多的IX型分泌蛋白(T9SS)，以及更多的阴离子A-内毒素，最后已知的是OM T9SS蛋白的系链。T9SS蛋白包含许多毒力蛋白，包括牙痛蛋白水解酶和 氨基精氨酸脱亚胺酶。因此，OMV被认为是毒力很强的长距离携带者。 有效载荷。由于OMV的脂质A分子已被证明是四酰化的，因此OMV是一种受欢迎的 T9SS毒力蛋白的环境，我们假设脱酰酶缺陷菌株的OM是， 相反，由于缺乏四酰化的脂类A，这些毒力因子会减弱。 鉴定了磷脂A脱酰基酶，该酶是磷脂A生物学中的一个关键步骤。 牙周炎，是我们之前的R21应用的重点。我们的工作导致了PGN_1123的鉴定，它是一种高度 保守基因，作为牙龈假单胞菌33277中的脱酰基酶编码基因。在本应用程序中，我们的目标是研究 脂蛋白A去酰化在牙周炎致病机制不同方面的作用 在目标1中，我们将比较野生型33277和∆pgn_1123突变体对油菜花叶病毒产量的影响。 A-内毒素、牙龈痛活性和炎性小体激活，这些都参与了牙龈假单胞菌的毒力。 在目的2中，我们将研究PGN_1123在疾病过程中的表达是否上调。 PGN_1123是三基因操纵子的最后一个基因。我们将比较这三个基因在体内的表达 该操纵子来自健康的和患病的牙龈下菌斑样本，这些样本来自人类受试者。 在∆pgn_1123突变体中毒力特性的降低之后将会有一个深入的 研究这种酶，以更好地了解脱酰化过程，并设计出抑制它的方法。增加了 PGN_1123在疾病过程中的表达可能暗示该基因受到调控。未来的研究将包括调查 它的调节，并评价其表达作为衡量疾病程度的诊断工具的使用。

Abstract The Gram-negative sub-gingival anaerobic bacterium Porphyromonas gingivalis is closely associated with periodontitis, a chronic inflammatory disorder. It is found in greater abundance in periodontal diseased sites than in healthy sites suggesting it is well equipped to survive hostile inflammatory conditions. In addition, mouse models of infection have demonstrated that infection with P. gingivalis can tilt the sub-gingival microbiome to a disease-inducing dysbiotic state. Lipid A, the hydrophobic anchor of lipopolysaccaharide, is the bacterial ligand of the host innate immune receptor, TLR4. In P. gingivalis, lipid A is modified by deacylation, from an initially synthesized penta- acylated structure to tetra-acylated structures, a step essential for evading the TLR4-mediated pro- inflammatory response. Tetra-acylated lipid A is also known to contribute significantly to bacterial virulence properties such as outer membrane vesicle (OMV) formation, and attenuation of inflammasome activation. OMV's in P. gingivalis are distinct from the outer membrane (OM) in that they contain more under- acylated lipid A, more Type IX secreted proteins (T9SS), and more anionic A-LPS, the last known to be an OM tether for T9SS proteins. T9SS proteins comprise many virulence proteins including gingipain proteases and peptidylarginine deiminase. OMVs, hence, are considered as long distance carriers of a heavy virulence payload. Since lipid A molecules of OMVs have been shown to be tetra-acylated, and OMVs are a favored environment for T9SS virulence proteins, we hypothesize that the OM of a deacylase deficient strain is, conversely, attenuated for these virulence factors due to absence of tetra-acylated lipid A. Identification of the as yet unknown lipid A deacylase, required for a key step in the lipid A biology of P. gingivalis, was the focus of our previous R21 application. Our work led to identification of PGN_1123, a highly conserved gene, as the deacylase-encoding gene in P. gingivalis 33277. In this application, our goal is to study the effect of lipid A deacylation on different aspects of P. gingivalis pathogenesis. In Aim 1, we will compare wild-type 33277 with the ∆PGN_1123 mutant for OMV production, amount of A-LPS, gingipain activity and inflammasome activation, all of which contribute to P. gingivalis virulence. In Aim 2, we will investigate whether expression of PGN_1123 is up-regulated during disease. PGN_1123 is the last gene of a three gene operon. We will compare in vivo expression of all three genes of the operon from healthy vs. diseased sub-gingival plaque samples obtained from human subjects. A decrease in potency of virulence properties in the ∆PGN_1123 mutant will be followed by an in depth study of the enzyme to better understand the deacylation process, and to devise ways of inhibiting it. Increased PGN_1123 expression during disease will imply the gene is regulated. Future studies will include investigating its regulation, and evaluating use of its expression as a diagnostic tool for measuring extent of disease.