Ambati Suresh, Pham Tuyetnhu, Lewis Zachary A, Lin Xiaorong, Meagher Richard B
Department of Genetics, University of Georgia, Athens, GA, 30602, USA.
Department of Microbiology, University of Georgia, Athens, GA, 30602, USA.
Fungal Biol Biotechnol. 2021 Dec 24;8(1):22. doi: 10.1186/s40694-021-00126-3.
Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans.
We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs.
DC-SIGN's CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.
危及生命的侵袭性真菌感染采用抗真菌药物进行治疗，如载有两性霉素B（AmB）的脂质体。我们在此的目标是表明，通过C型凝集素病原体识别受体DC-SIGN将载有AmB的脂质体靶向真菌细胞可提高抗真菌活性。DC-SIGN可结合多种交联的富含甘露糖和岩藻糖基化的聚糖以及脂甘露聚糖，这些物质由包括三种最具生命威胁的真菌烟曲霉、白色念珠菌和新型隐球菌在内的蠕虫、原生生物、真菌、细菌和病毒病原体表达。人DC-SIGN的配体识别由与膜转运和信号序列相连的碳水化合物识别结构域（CRD）提供。不同蛋白质异构体中表达的八个颈部重复序列（NR1至NR8）的不同组合可能会改变CRD的方向，以增强其与不同聚糖的结合。
我们制备了两种重组异构体，在异构体DCS12中将CRD与NR1和NR2结合，在异构体DCS78中将CRD与NR7和NR8结合，并将它们偶联到脂质载体上。将这些构建体插入载有聚乙二醇化AmB的脂质体AmB-LLs的膜中，以产生DCS12-AmB-LLs和DCS78-AmB-LLs。相对于AmB-LLs和牛血清白蛋白包被的BSA-AmB-LLs，DCS12-AmB-LLs和DCS78-AmB-LLs在体外与烟曲霉、白色念珠菌和新型隐球菌产生的胞外多糖基质的结合更有效，其中DCS12-AmB-LLs的表现优于DCS78-AmB-LLs。DCS12-AmB-LLs在体外对所有这三种菌种的抑制和/或杀灭作用明显优于AmB-LLs或BSA-AmB-LLs。在侵袭性念珠菌病和肺曲霉病的小鼠模型中，一剂低剂量的DCS12-AmB-LLs相对于AmB-LLs分别使肾脏和肺部的真菌负荷显著降低了几倍。
DC-SIGN的CRD将抗真菌脂质体特异性靶向三种进化上高度不同的致病真菌，并在体外和体内增强了载有AmB的脂质体的抗真菌功效。靶向作用显著降低了抗真菌药物的有效剂量，这可能降低药物毒性，有效克服剂量依赖性耐药性，并更有效地杀死持续存在的细胞。除了真菌外，DC-SIGN靶向脂质体包装的抗感染药物有可能改变针对来自不同界的多种病原体（包括表达其同源配体的原生动物、蠕虫、细菌和病毒）的治疗模式。
