Interaction of polymicrobial biofilms with human immune cells

多药耐药性正在导致整个抗生素种类的冗余，并使“抗生素时代”的终结成为可能。在“抗生素时代”，常见的感染和轻微的伤害都可能导致死亡。ESKAPE病原菌（粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌科）对抗生素的杀生物作用具有耐药性，因此迫切需要在更好地了解ESKAPE病原菌致病性的基础上开发新的抗菌药物。绿脓杆菌（aeruginosa， PA）是一种用途广泛的环境生物。PA盛行于医院环境中，在免疫功能低下和烧伤患者中引起严重感染。PA感染在囊性纤维化（CF）患者中尤其麻烦，因为它是肺功能不可逆转丧失和死亡率的主要决定因素。由于其对抗生素的内在抗性和形成生物膜的能力，破坏免疫防御使PA能够引起顽固性感染。从浮游到无根生长和生物膜发育的转变是PA发病机制的核心。目前迫切需要更好地了解转向生物膜生活方式如何促进PA毒性以及免疫细胞如何检测和响应这些结构。PA生物膜由含有两种主要碳水化合物聚合物Psl和Pel的细胞外基质组成。我们最近的研究结果表明，免疫细胞表达的三种主要的碳水化合物结合（凝集素）受体DC-SIGN (CD209)，甘露糖受体（MR, CD206）和Dectin-2与PA生物膜相互作用。结合是钙依赖的，被相关糖抑制，并在PAO1和临床分离物形成的生物膜中观察到。DC-SIGN、MR和Dectin-2配体在生物膜内的分布存在差异。人单核细胞来源的树突状细胞（huDCs）与含有Psl+/Pel+、Psl+/Pel-和Psl-/Pel+生物膜的孔一起培养，与含有不能形成生物膜的细菌的孔一起培养，Psl-/Pel-选择性地产生IL- 1b（4和18小时）和IL-23（仅4小时），与含有生物膜的孔相比，含有更多Psl的生物膜倾向于诱导较少的IL-23。此外，huDCs中凝集素受体的特异性阻断改变了它们对PA生物膜的反应。这些结果表明，生物膜不仅提供了抵抗免疫攻击的有效屏障，而且还可以通过参与凝集素受体来调节PA感染背景下的免疫细胞激活。许多感染，如CF患者的肺部感染，本质上是多微生物的。大多数CF患儿的呼吸道最初都有金黄色葡萄球菌。随着时间的推移，金黄色葡萄球菌通常被PA取代，导致一段时间的合并感染。金黄色葡萄球菌可以在糖萼或黏液层内产生多层生物膜。糖杯含有多糖抗原，称为多糖细胞间抗原（PIA）。PIA由b -1,6连接的n-乙酰氨基氨基残基（80-85%）和含有磷酸盐和酯连接的琥珀酸盐（15-20%）的非n-乙酰化d -氨基残基含量较低的阴离子部分组成。迄今为止还没有发现PIA的受体。我们假设由PA和金黄色葡萄球菌形成的混合生物膜将显示改变的碳水化合物产生，导致凝集素受体的不同参与和人类免疫细胞的不同激活。该项目的具体目标是：(i)研究PA和金黄色葡萄球菌混合生物膜如何影响人体免疫细胞的激活（ii）并确定凝集素受体对这些反应的贡献。

Multidrug resistance is causing redundancy of entire classes of antibiotics and making the end of the "antibiotic era", in which common infections and minor injuries can kill, a real possibility. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) are resistant to the biocidal action of antibiotics and there is an urgent need for the development of new antimicrobial options underpinned by a better understanding of the pathogenicity of ESKAPE organisms.P. aeruginosa (PA) is a highly versatile environmental organism. PA flourishes in the hospital setting where it causes serious infections in immunocompromised and burn patients. PA infection is especially troublesome in Cystic Fibrosis (CF) patients where it is a major determinant of the irreversible loss of lung function and mortality. Breach of immune defences enables PA to cause intractable infections due to its intrinsic resistance to antibiotics and ability to form biofilms. Transition from planktonic to sessile growth and biofilm development are central to PA pathogenesis. There is an urgent need to better understand how the switch to a biofilm life style contributes to PA virulence and how immune cells detect and respond to these structures. PA biofilms consist of an extracellular matrix containing two main carbohydrate polymers, Psl and Pel. Our recent results show that three major carbohydrate-binding (lectin) receptors expressed by immune cells, DC-SIGN (CD209), the mannose receptor (MR, CD206) and Dectin-2, interact with PA biofilms. Binding is calcium-dependent, inhibited by relevant sugars, and observed in biofilms formed by PAO1 and clinical isolates. DC-SIGN, MR and Dectin-2 ligands within biofilms showed differential distribution. Human monocyte-derived dendritic cells (huDCs) incubated with wells containing Psl+/Pel+, Psl+/Pel- and Psl-/Pel+ biofilms alongside wells containing bacteria non capable of biofilm formation Psl-/Pel- selectively produce IL-1B (4 and 18 h) and IL-23 (only 4 h) in response to biofilm-containing wells, with biofilms containing more Psl tending to induce less IL 23. Further, specific blockage of lectin receptors in huDCs alters their response to PA biofilms. These results suggest that biofilms do not just provide an effective barrier against immune attack but could also modulate immune cell activation in the context of PA infection through engagement of lectin receptors. Many infections, such as pulmonary infections occurring in patients with CF, are polymicrobial in nature. Most children with CF are initially colonised with S. aureus in their airways. With time S. aureus is commonly replaced with PA, resulting in a period of co-infection. S. aureus can produce a multilayered biofilm embedded within a glycocalyx or slime layer. The glycocalix contains a polysaccharide antigen named polysaccharide intercellular antigen (PIA). PIA is composed of B-1,6-linked N-acetylglucosamine residues (80-85%) and an anionic fraction with a lower content of non-N-acetylated D-glucosaminyl residues that contains phosphate and ester-linked succinate (15-20%). No receptors for PIA have been described to date. We hypothesise that mixed biofilms formed by PA and S. aureus will display altered carbohydrate production leading to differential engagement of lectin receptors and differential activation of human immune cells. The specific aims of this project are (i) to investigate how PA and S. aureus mixed biofilms influence the activation of human immune cells (ii) and determine the contribution of lectin receptors to these responses.