State Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin Province, People's Republic of China.
Jiangsu Key Laboratory of Marine Bioresources and Environment, Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang, 222005, Jiangsu Province, People's Republic of China.
Parasit Vectors. 2023 Mar 3;16(1):85. doi: 10.1186/s13071-023-05688-2.
Giardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated.
Recombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1β, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1β secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice.
Alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1β and pro-caspase-1, significant enhancement of IL-1β secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1β secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice.
Overall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.
蓝氏贾第鞭毛虫是一种寄生虫，可引起贾第虫病，这是一种肠道感染，尤其常见于幼儿，临床症状为腹泻。我们之前曾报道过，细胞外的蓝氏贾第鞭毛虫可触发细胞内核苷酸结合寡聚化结构域样受体 3（NLRP3）炎症小体的激活，并通过分泌细胞外囊泡（EVs）来调节宿主炎症反应。然而，在这个过程中，蓝氏贾第鞭毛虫 EVs（GEVs）中涉及的确切病原体相关分子模式以及 NLRP3 炎症小体在贾第虫病中的作用仍有待阐明。
构建了重组真核表达质粒 pcDNA3.1(+)-alpha-2 和 alpha-7.3 贾第虫在 GEVs 中的表达，转染原代小鼠腹腔巨噬细胞，并通过测量炎症小体靶分子半胱天冬酶-1 p20 的表达水平进行筛选。通过测量 NLRP3 炎症小体（NLRP3、前白细胞介素-1β[IL-1β]、前半胱天冬酶-1 和半胱天冬酶-1 p20）的关键分子的蛋白表达水平、IL-1β 的分泌水平、凋亡斑点样蛋白（ASC）寡聚化的水平以及 NLRP3 和 ASC 的免疫荧光定位，进一步验证了蓝氏贾第鞭毛虫 alpha-2 和 alpha-7.3 贾第虫的初步鉴定。然后，使用 NLRP3 激活被阻断的（NLRP3 阻断小鼠）小鼠来评估 NLRP3 炎症小体在蓝氏贾第鞭毛虫致病性中的作用，并监测体重、十二指肠中的寄生虫负担和十二指肠的组织病理学变化。此外，我们还探讨了 alpha-2 和 alpha-7.3 贾第虫是否通过 NLRP3 炎症小体在体内引发 IL-1β 分泌，并确定这些分子在小鼠中对蓝氏贾第鞭毛虫致病性的作用。
alpha-2 和 alpha-7.3 贾第虫在体外触发 NLRP3 炎症小体的激活。这导致半胱天冬酶-1 p20 的激活，NLRP3、前白细胞介素-1β和前半胱天冬酶-1 的蛋白表达水平上调，IL-1β 的分泌显著增强，细胞质中 ASC 斑点的形成，以及 ASC 寡聚化的诱导。NLRP3 炎症小体的缺失加重了蓝氏贾第鞭毛虫在小鼠中的致病性。与用包囊灌胃的野生型小鼠相比，用包囊灌胃的 NLRP3 阻断小鼠中的滋养体负荷增加，十二指肠绒毛损伤严重，表现为坏死隐窝伴萎缩和分支。体内实验表明，alpha-2 和 alpha-7.3 贾第虫可通过 NLRP3 炎症小体诱导 IL-1β 分泌，而 alpha-2 和 alpha-7.3 贾第虫的免疫接种可降低小鼠中的蓝氏贾第鞭毛虫致病性。
总的来说，本研究的结果表明，alpha-2 和 alpha-7.3 贾第虫可触发宿主 NLRP3 炎症小体的激活，并降低小鼠中蓝氏贾第鞭毛虫的感染能力，这为预防贾第虫病提供了有希望的靶点。
