A zebrafish leprosy model identifies interferon and plasminogen signaling as survival determinants in mycobacterial infection

Although many genes are associated with leprosy, a skin and nerve infection by Mycobacterium leprae, the function of most of these genes in infection remains unknown. This is partly due to a paucity of animal models that are genetically malleable and recapitulate features of the human disease. Zebrafish, a recent leprosy model, have human-like responses to M. leprae, including macrophage-mediated inflammation and neurodegeneration. We confirm this at the transcriptional level, using RNA sequencing (RNAseq) of chronic M. leprae infection of adult zebrafish. This identified regulated zebrafish orthologs of human leprosy-associated genes, including {beta}- and {gamma}-interferon, IL-6, IL-10, IL-4, and TNF superfamily members. Other regulated genes have not been previously associated with leprosy. Genes associated with tuberculoid leprosy (T-lep) were largely downregulated, while lepromatous leprosy (L-lep) genes were upregulated. Pathways relevant to leprosy, such as phagocytosis and antiviral responses, differed between early and late stages of infection. In infected rag1 mutant zebrafish, which lack T and B cells, T-lep genes were downregulated, suggesting that adaptive immunity is required for their expression. Key pathways and genes were validated by qPCR using zebrafish infection with M. marinum:PGL-1, a model pathogen that can express M. leprae genes. This system allowed for the identification of validated M. leprae-regulated genes that alter infection outcomes, by using multiplex CRISPR to simultaneously mutate many zebrafish genes. 17 genes were screened and 2 were identified that increased mortality infection: isg15 (interferon-stimulated gene 15) and serpine1 (plasminogen activator inhibitor-1). This work confirms that, in humans and zebrafish, type I interferon and plasminogen activation are required for survival of mycobacterial disease, and demonstrates that multiplex CRISPR can identify mediators of host defense in vivo.