LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50.
LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50.
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DOI：
10.1128/jb.00751-16
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发表时间：
2017-06-01
影响因子：
3.2
通讯作者：
Curtis MA
中科院分区：
文献类型：
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作者：
Rangarajan M;Aduse-Opoku J;Hashim A;McPhail G;Luklinska Z;Haurat MF;Feldman MF;Curtis MA
牙龈卟啉单胞菌产生富含毒力因子的外膜囊泡 (OMV)，包括半胱氨酸蛋白酶和 A-LPS（该生物体产生的两种脂多糖 (LPS) 之一）。先前的研究表明，A-LPS 和 PG0027（一种外膜 (OM) 蛋白）可能参与 OMV 的形成。使用 W50 亲本和 ΔPG0027 突变株检查了它们在此过程中的作用。 PG0027 失活导致 OMV 产量减少。通过基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF MS) 分析牙龈卟啉单胞菌 W50 和 ΔPG0027 突变株的细胞和 OMV 中的脂质 A。 W50细胞中的脂质A含有双-P-五酰基、单-P-五酰基、单-P-四酰基、非-P-五酰基和非-P-四酰基物质，而来自ΔPG0027突变细胞的脂质A仅含有磷酸化物质；不存在非磷酸化物种。来自 ΔPG0027 的单 P-五酰基 (m/z 1,688) 和单 P-四酰基 (m/z 1,448) 脂质 A 的 MALDI-TOF/TOF 串联 MS 显示两者都含有脂质 A 1-磷酸，表明 ΔPG0027 突变株缺乏脂质 A 1-磷酸酶活性。 W50和ΔPG0027突变株的总磷酸酶活性相似，而ΔPG0027突变株周质中的磷酸酶活性低于W50，支持PG0027在脂质A去磷酸化中的作用。 W50 OMV 富含 A-LPS，其脂质 A 不含非磷酸化物质，而 ΔPG0027 突变体（OMV 和细胞）的脂质 A 含有相似的物质。因此，牙龈卟啉单胞菌中的OMV显然是在富含A-LPS且不含非磷酸化脂质A的OM区域中形成的。相反，为了优化OMV的形成，需要通过PG0027依赖性过程对脂质A进行去磷酸化。因此，非磷酸化和磷酸化脂质 A 的相对比例似乎对该生物体中 OMV 的形成至关重要。重要性 革兰氏阴性细菌通过外膜 (OM) 的“起泡”产生外膜囊泡 (OMV)。 OMV 可以进攻性地用作毒力因子的传递系统，也可以防守性地帮助宿主定植和细菌在恶劣环境中的生存。早期以口腔厌氧菌牙龈卟啉单胞菌为模式生物研究OMV形成机制的研究表明，OM蛋白PG0027和该生物合成的两种脂多糖(LPS)之一，即A-LPS，在OMV形成中发挥重要作用。我们提出了牙龈卟啉单胞菌中OMV形成的新机制，涉及由PG0027控制/调节的A-LPS的脂质A的去磷酸化，这导致OM不稳定，导致起泡和OMV的产生。
Porphyromonas gingivalis produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z 1,688) and mono-P-tetraacyl (m/z 1,448) lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism. IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.