Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.
Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.
复制标题
DOI：
10.1074/jbc.m114.566570
复制
发表时间：
2014-09-05
期刊：
影响因子：
--
通讯作者：
Gerke C
中科院分区：
文献类型：
--
作者：
Rossi O;Pesce I;Giannelli C;Aprea S;Caboni M;Citiulo F;Valentini S;Ferlenghi I;MacLennan CA;D'Oro U;Saul A;Gerke C
背景：来自革兰氏阴性菌的GMMA是一种有吸引力的疫苗技术，但脂多糖（LPS）的反应原性限制了使用。结果：导致五酰化的遗传LPS修饰将志贺菌GMMA反应原性降低至TLR 2介导的限度。导致棕榈油酰化六酰化LPS的修饰引发更高的TLR 4介导的反应原性。结论：使用GMMA作为疫苗可能需要LPS五酰化。意义：了解TLR激活的相对贡献指导GMMA疫苗开发。来自革兰氏阴性菌的外膜颗粒是有吸引力的疫苗候选物，因为它们在其天然环境中呈递表面抗原。我们以前开发了一种高产量的生产工艺，用于生产志贺氏菌的遗传衍生颗粒，称为膜抗原通用模块（GMMA）。由于GMMA来源于外膜，它们含有免疫刺激成分，特别是脂多糖（LPS）。我们研究了通过修饰脂A，LPS的内毒素部分，通过删除晚期酰基转移酶基因，msbB或htrB，在产生GMMA的宋内志贺菌和福氏志贺菌菌株中降低其反应原性的方法。来自msbB突变体的具有所得五酰化脂质A的GMMA显示出降低600倍的能力，而来自S. Sonnei ΔhtrB突变体在报告细胞系中显示出与具有野生型脂质A的GMMA相比降低60，000倍的刺激人Toll样受体4（TLR 4）的能力。在人外周血单核细胞中，GMMA与五酰化脂质A在诱导炎性细胞因子方面显示出显著降低（S。sonnei ΔhtrB，800倍; ΔmsbB突变体，300倍）。我们发现，这些GMMA的残留活性主要是由于非脂质A相关的TLR 2激活。相反，在S. flexneri ΔhtrB突变体，一种补偿性脂质A棕榈油酰化导致具有六酰化脂质A的GMMA比具有五酰化脂质A的GMMA具有高约10倍的刺激外周血单核细胞的活性，主要是由于保留的TLR 4活性。因此，为了用作疫苗，GMMA可能需要脂质A五酰化。结果确定了GMMA激活TLR 4和TLR 2的相对贡献，这需要考虑GMMA疫苗的开发。
Background: GMMA from Gram-negative bacteria are an attractive vaccine technology, but lipopolysaccharide (LPS) reactogenicity limits use. Results: Genetic LPS modification resulting in penta-acylation reduced Shigella GMMA reactogenicity to a TLR2-mediated limit. Modifications resulting in palmitoleoylated hexa-acylated LPS triggered higher TLR4-mediated reactogenicity. Conclusion: Use of GMMA as vaccines will likely require LPS penta-acylation. Significance: Understanding the relative contribution of TLR activation guides GMMA vaccine development. Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.