Alaei Sarah R, King Alisa J, Banani Karim, Reddy Angel, Ortiz Joshua, Knight Alexa L, Haldeman Jessica, Su Thet Hnin, Park Hana, Coats Stephen R, Jain Sumita
Division of Science and Mathematics, School of Interdisciplinary Arts and Sciences, University of Washington, Tacoma, Washington, USA.
Department of Periodontics, School of Dentistry, University of Washington, Seattle, Washington, USA.
J Bacteriol. 2025 Jan 31;207(1):e0033624. doi: 10.1128/jb.00336-24. Epub 2024 Dec 11.
Outer membrane vesicles (OMVs) are small membrane enclosed sacs released from bacteria which serve as carriers of biomolecules that shape interactions with the surrounding environment. The periodontal pathogen, , is a prolific OMV producer. Here, we investigated how the structure of lipid A, a core outer membrane molecule, influences OMV production, OMV-dependent TLR4 activation, and biofilm formation. We examined mutant strains of 33277 deficient for enzymes that alter lipid A phosphorylation and acylation status. The lipid A C4'-phosphatase ()-deficient strain and strains bearing inactivating point mutations in the LpxF active site displayed markedly reduced OMV production relative to WT. In contrast, strains deficient for either the lipid A C1-phosphatase () or the lipid A deacylase (PGN_1123; ) genes did not display alterations in OMV abundance compared to WT. These data indicate that lipid A C4'-phosphate removal is required for typical OMV formation. In addition, OMVs produced by and strains, possessing only penta-acylated lipid A, stimulated robust TLR4 activation, whereas OMVs obtained from WT and strains, containing predominantly tetra-acylated lipid A, did not. Hence, lipid A remodeling modulates the capacity of OMVs to engage host TLR4-dependent immunity. Finally, we demonstrate an inverse relationship between OMV abundance and biofilm density, with the mutants forming denser biofilms than either WT, , or strains. Therefore, OMVs may also contribute to pathogenesis by regulating biofilm formation and dispersal.IMPORTANCE is a bacterium strongly associated with periodontitis. exports lipids, proteins, and other biomolecules that contribute to the bacterium's ability to persist in inflammatory conditions encountered during disease. These biomolecules are exported through several mechanisms, including via outer membrane vesicles (OMVs). Despite their ubiquity, the mechanisms that drive outer membrane vesicle production vary among bacteria and are not fully understood. In this study, we report that C4' dephosphorylation of lipid A, a major outer membrane molecule, is required for robust outer membrane vesicle production and biological function in . This finding adds to the growing body of evidence that lipid A structure is an important factor in outer membrane vesicle biogenesis in diverse bacterial species.
外膜囊泡（OMV）是从细菌释放的小的膜包裹囊泡，作为生物分子的载体，塑造与周围环境的相互作用。牙周病原体牙龈卟啉单胞菌是一种多产OMV的细菌。在这里，我们研究了核心外膜分子脂多糖A的结构如何影响OMV的产生、OMV依赖的Toll样受体4（TLR4）激活以及生物膜形成。我们检测了牙龈卟啉单胞菌33277中缺乏改变脂多糖A磷酸化和酰化状态的酶的突变菌株。与野生型相比，脂多糖A C4'-磷酸酶（LpxH）缺陷菌株和在LpxF活性位点带有失活点突变的菌株显示出OMV产生显著减少。相比之下，脂多糖A C1-磷酸酶（LpxP）或脂多糖A脱酰酶（PGN_1123；LpxL）基因缺陷的菌株与野生型相比，OMV丰度没有变化。这些数据表明典型的OMV形成需要去除脂多糖A的C4'-磷酸。此外，仅具有五酰化脂多糖A的牙龈卟啉单胞菌和LpxL突变体菌株产生的OMV刺激了强烈的TLR4激活，而从主要含有四酰化脂多糖A的野生型和LpxF突变体菌株获得的OMV则没有。因此，脂多糖A重塑调节了OMV参与宿主TLR4依赖性免疫的能力。最后，我们证明了OMV丰度与生物膜密度之间呈负相关，LpxH突变体形成的生物膜比野生型、LpxF或LpxL菌株更致密。因此，OMV也可能通过调节生物膜的形成和扩散来促进发病机制。重要性牙龈卟啉单胞菌是一种与牙周炎密切相关的细菌。它输出脂质、蛋白质和其他生物分子，这些分子有助于该细菌在疾病期间遇到的炎症条件下持续存在。这些生物分子通过多种机制输出，包括通过外膜囊泡（OMV）。尽管它们无处不在，但驱动外膜囊泡产生的机制在细菌之间各不相同，尚未完全了解。在这项研究中，我们报告脂多糖A（一种主要的外膜分子）的C4'去磷酸化是牙龈卟啉单胞菌中强大的外膜囊泡产生和生物学功能所必需的。这一发现增加了越来越多的证据，即脂多糖A结构是不同细菌物种中外膜囊泡生物发生的一个重要因素。
