Supplementary Material for: Macrophage-Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti-Bacterial Function in Zebrafish

Macrophage-expressed gene 1 (MPEG1) encodes an evolutionarily
conserved protein with a predicted membrane attack complex/perforin
domain associated with host defence against invading pathogens. In
vertebrates, MPEG1/perforin-2 is an integral membrane protein of
macrophages, suspected to be involved in the killing of intracellular
bacteria by pore-forming activity. Zebrafish have 3 copies of MPEG1; 2 are expressed in macrophages, whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is downregulated during infection with both pathogens, mpeg1.2 is infection inducible. Upregulation of mpeg1.2
is partially dependent on the presence of functional Mpeg1 and requires
the Toll-like receptor adaptor molecule MyD88 and the transcription
factor NFκB. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in an increased bacterial burden. In Salmonella typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase the bacterial burdens, but mpeg1
morphants show increased survival times. The combined results of these
two in vivo infection models support the anti-bacterial function of the
MPEG1/perforin-2 family and indicate that the intricate cross-regulation
of the two mpeg1 copies aids the zebrafish host in combatting infection of various pathogens.

巨噬细胞表达基因1（Macrophage-expressed gene 1, MPEG1）编码一种进化保守的蛋白质，其携带有与宿主抵御入侵病原体的免疫防御功能相关的预测性膜攻击复合物/穿孔素结构域（membrane attack complex/perforin domain）。在脊椎动物（vertebrates）中，MPEG1/穿孔素2（perforin-2）是巨噬细胞（macrophages）的整合膜蛋白，被认为可通过成孔活性杀伤胞内细菌（intracellular bacteria）。斑马鱼（Zebrafish）拥有3个MPEG1拷贝：其中2个在巨噬细胞中表达，第三个则可能为假基因（pseudogene）。mpeg1与mpeg1.2基因在斑马鱼胚胎（zebrafish embryos）感染海分枝杆菌（Mycobacterium marinum）和鼠伤寒沙门氏菌（Salmonella typhimurium）这两种细菌性病原体时呈现差异调控模式：mpeg1在两种病原体感染下均被下调，而mpeg1.2则为感染诱导型基因。mpeg1.2的上调部分依赖于功能性Mpeg1的存在，且需要Toll样受体衔接分子MyD88（Toll-like receptor adaptor molecule MyD88）以及转录因子NF-κB（transcription factor NFκB）的参与。敲低mpeg1会改变宿主对海分枝杆菌感染的免疫应答，并导致细菌载量升高。在鼠伤寒沙门氏菌感染模型中，同时敲低mpeg1与mpeg1.2均会提升细菌载量，但mpeg1基因敲低个体的存活时间反而延长。上述两种体内感染模型的综合结果证实了MPEG1/穿孔素2家族的抗菌功能，并表明两个mpeg1拷贝之间的复杂交叉调控可帮助斑马鱼宿主对抗多种病原体的感染。