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

Macrophage-expressed gene 1 <i>(MPEG1)</i> 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 <i>MPEG1</i>; 2 are expressed in macrophages, whereas the third could be a pseudogene. The <i>mpeg1</i> and <i>mpeg1.2</i> genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens <i>Mycobacterium marinum</i> and <i>Salmonella typhimurium.</i> While <i>mpeg1</i> is downregulated during infection with both pathogens, <i>mpeg1.2</i> is infection inducible. Upregulation of <i>mpeg1.2</i> 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 <i>mpeg1</i> alters the immune response to <i>M. marinum</i> infection and results in an increased bacterial burden. In <i>Salmonella typhimurium</i> infection, both <i>mpeg1</i> and <i>mpeg1.2</i> knockdown increase the bacterial burdens, but <i>mpeg1</i> 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 <i>mpeg1</i> copies aids the zebrafish host in combatting infection of various pathogens.