Tuberculosis (TB) is estimated to be responsible for a third to a half of all mortality in HIV-infected patients since the beginning of the AIDS epidemic. Current treatments of TB/HIV co-infection remain complicated by complex drug regimens, drug-drug interactions and toxicity, and drug resistance. Thus, novel treatment approaches to enhance therapy are of critical importance for advances in the control of TB and TB/HIV co-infection. To establish infection, Mycobacterium tuberculosis (MTb) and HIV must evade host innate immune mechanisms including those that detect pathogen-derived nucleic acids. Our recent discoveries have shown that molecules in the type I interferon (IFN) and RNA sensing pathways are critical host factors that restrict MTb growth in human cells. Furthermore, we have shown that that the FDA- approved drug nitazoxanide (NTZ) significantly enhances type I interferon (IFN) and RNA sensing pathways, induces the cell stress gene GADD34, and inhibits intracellular MTb growth. Our preliminary data show that NTZ significantly inhibits intracellular growth of HIV, MTb, and HIV/TB co-infection in primary human cells (PBMC, monocyte-derived-macrophages (MDM), and monocytes) and in a model we have established in a human pulmonary epithelial cell line using infectious isolates of HIV and MTb. Furthermore, using CRISPR- edited or shRNA modified cells that are deficient in IFITM3 or the type I IFN receptor (IFNAR) and imaging flow cytometry we have optimized for evaluation of MTb and HIV growth, we have shown that IFITM3 and IFN signaling are critical to innate control of both MTb and HIV growth. Based on these preliminary data, we will test here the overarching hypothesis that an overlapping set of innate immune molecules that intersect the RNA sensing, type I IFN, and cell stress signaling pathways are at the intersection of control of TB and HIV growth and are responsible for NTZ’s ability to inhibit both pathogens. In Aim 1, we will use CRISPR targeting to test the role of key RNA sensor and IFN signaling molecules in host control of HIV and TB/HIV infections in our TB/HIV models and primary MDM. In parallel, we will establish a novel human lung alveolar epithelial (AT2) cell organoid for study of TB, HIV, and TB/HIV co-infection using single-cell-RNA-seq to interrogate transcriptomes of infected cells and bystander cells to test the hypothesis that MTb-infected cells and NTZ significantly influence the growth of MTb-uninfected/HIV infected cells and the infectability of naïve bystander cells. In Aim 3, we will combine unbiased RNA-seq, CRISPR screening, and profiling of NTZ- induced phosphorylation of major signaling pathways to test our hypothesis that NTZ inhibits TB and HIV via a set of molecules whose function converges in the IFN and cell stress pathways. Together, these experiments will identify genes and molecules that inhibit TB and HIV, establish a new organoid model of TB and HIV infection, identify novel targets for host-direc...