The necrosis-inducing toxin of Mycobacterium tuberculosis

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 DESCRIPTION (provided by applicant): Tuberculosis (TB) is one of the most important and widespread bacterial diseases and a global health threat. Survival within infected macrophages is a key feature of Mycobacterium tuberculosis (Mtb) pathogenesis and is crucial to a persistent infection in the human host. The lack of any protein with sequence similarities to bacterial toxins and the apparent absence of a protein toxin with profound effects on host cells led to the widely accepted assumption that Mtb does not have these classical virulence factors. However, this paradigm was challenged by our discovery of the outer membrane protein CpnT which is utilized by Mtb to secrete a toxic C- terminal domain. This toxin is required by Mtb to replicate i macrophages, is the major cytotoxicity factor of Mtb in macrophages and causes necrotic cell death of host cells. Hence, we named this protein Tuberculosis Necrotizing Toxin (TNT). Killing of infected macrophages by necrosis is a well-known, but poorly understood virulence mechanism of Mtb to escape phagosomal containment and the adaptive immune response. Recently, we discovered that TNT is a novel NAD+-glycohydrolase which gains access to the cytosol of infected macrophages in an ESX-1 dependent manner. Mtb protects itself from CpnT/TNT toxicity by producing an antitoxin which we named Immunity Factor of TNT (IFT). IFT inactivates the enzyme activity of TNT upon binding. The discovery of IFT enabled us to produce TNT protein in mg quantities and, subsequently, to solve the crystal structure of the TNT-IFT complex and characterize the purified TNT protein enzymatically. We have identified catalytically inactive TNT mutants, which we will use to define the role of this Mtb toxin in the molecular pathway ultimately leading to necrosis of infected macrophages. Thus, we are now able to examine a novel molecular mechanism of paramount importance for the interaction of Mtb with host cells. These findings will also be important for more than 200 bacterial and fungal pathogens with TNT homologs and for understanding the molecular consequences of NAD+ depletion in host cells.