Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

Abstract

222 SUMMARY: 23 Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death 24 worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating 25 the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated 26 high-throughput genetic screens to identify perturbations that improve the survival of 27 human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-28 Gué rin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations 29 constrained the growth of intracellular mycobacteria. We identified over 100 genes 30 associated with diverse biological pathways as potential HDT targets. We validated key 31 components of the type I interferon and aryl hydrocarbon receptor signaling pathways that 32 respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these 33 inhibitors enhanced human macrophage survival and limited the intracellular growth of 34 Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex host-35 pathogen interactions and serve to identify HDTs with the potential to improve TB 36 treatment. 37 38

High-throughput CRISPR screens to dissect macrophage-Shigellainteractions

“…The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available on Addgene #92385). Both secondary CRISPR knockout and CRISPRi libraries, with 10 sgRNAs per gene, were designed to target the 251 genes scored in primary genome-wide screens (133 of these genes were identified as being involved in S. flexneri infection) and 121 genes from literature (47 of these genes were found to be involved in S. flexneri infection) 21 . 1000 non-targeting sgRNAs were used as controls 21 .…”

“…Monoclonal Cas9 and dCas9-Krab-expressing THP-1 cell lines were constructed and transduced with lentiviral sgRNA libraries 21 . High coverage CRISPR knockout and CRISPRi libraries were split for subsequent S. flexneri infection.…”

“…Genome-wide genetic hits were identified by comparing sgRNA abundance between infected samples and non-infected controls. Based on those host targets, secondary CRISPR knockout and CRISPRi screen libraries were designed and prepared 21 . Similarly, secondary positive screens were performed to validate those host targets.…”

“…Such infection with S. flexneri can be utilized as selective pressure for subsequent host survival-based genetic screens. Independent biological triplicates of genome-wide CRISPR knockout and CRISPRi screen libraries were prepared in THP-1 cells expressing Cas9 and dCas9-Krab, respectively 19–21 (Extended Data Fig. 2).…”

“…We next designed and prepared secondary CRISPR knockout and CRISPRi screen libraries targeting 372 human genes in order to validate genome-wide screen hits 21 , to test genes that were associated with different types of host cell death, and to compare the performance between CRISPR knockout and knockdown libraries by ensuring that there were consistent numbers of sgRNAs per gene in each type of library. Similar to our genome-wide screens, surviving THP-1 cells were harvested following S. flexneri infection, and the results of the screens were visualized with volcano plots (Extended Data Fig.…”