Interaction of polymicrobial biofilms with human immune cells

多重耐药性正在导致整个抗生素类别的冗余，并使“抗生素时代”的结束成为可能，在这个时代，常见的感染和轻微的伤害可能会导致死亡。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细菌对抗生素的杀灭作用具有耐药性，因此迫切需要开发新的抗菌药物，以更好地了解ESKAPE微生物的致病性。PA在医院环境中蓬勃发展，在那里它会导致免疫功能低下和烧伤患者的严重感染。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的主要决定因素。由于PA对抗生素的内在耐药性和形成生物膜的能力，免疫防御系统的破坏使其能够引起难治性感染。从浮游到固着生长的过渡和生物膜的发育是PA发病机制的核心。迫切需要更好地了解转变为生物膜的生活方式如何有助于PA的毒力，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糖结合受体：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，被相关的糖抑制，并在PAO1和临床分离株形成的生物膜中观察到。DC-SIGN、MR和Dectin-2配体在生物膜内呈不同分布。人单核细胞来源的树突状细胞(HuDC)与含有PSL+/Pel+、PSL+/Pel-和PSL-/Pel+生物膜的孔一起孵育，与含有不能形成生物膜的细菌的孔一起孵育，选择性地产生IL-1B(4和18h)和IL-23(仅4h)，含有较多PSL的生物膜倾向于产生较少的IL-23。此外，HuDC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，而且还可以通过凝集素受体的参与来调节PA感染背景下的免疫细胞激活。许多感染，如发生在CF患者身上的肺部感染，本质上是多菌的。大多数患有CF的儿童最初在他们的呼吸道中带有金黄色葡萄球菌。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产生多层生物膜，嵌入糖萼或粘液层中。糖杯含有一种名为多糖胞间抗原(PIA)的多糖抗原。PIA由B-1，6-连接的N-乙酰氨基葡萄糖残基(80%-85%)和含有磷酸盐和酯连接的丁二酸酯的非N-乙酰化D-氨基葡萄糖残基含量较低的阴离子部分组成(15%-20%)。到目前为止，还没有PIA的受体被描述。我们推测，金黄色葡萄球菌和PA形成的混合生物膜将显示碳水化合物产生的改变，导致凝集素受体的不同结合和人类免疫细胞的不同激活。本项目的具体目标是(I)研究金黄色葡萄球菌和PA混合生物膜如何影响人类免疫细胞的激活(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.