All ETDs from UAB

Advisory Committee Chair

Michael Niederweis

Advisory Committee Members

Peter Prevelige

Terje Dokland

Jamil Saad

William Benjamin

Document Type

Dissertation

Date of Award

2020

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Mycobacterium tuberculosis (Mtb) is a highly infectious airborne bacterial pathogen, and the causative agent of human tuberculosis. Mtb infects approximately one-third of the human population, and is responsible for more deaths than any other infectious disease in the history of mankind. Mtb is an intracellular pathogen and primarily infects myeloid cells, such as macrophages in the human lung. In macrophages, Mtb establishes a replication niche within membrane-bound vacuoles named phagosomes, while subverting immunity. The intracellular survival of Mtb depends on manipulation of host cellular processes through secreted effector proteins. Ultimately, this intracellular vacuole is ruptured by the type VII secretion system and Mtb translocates to the host cytosol. During this stage Mtb induces a necrotic-like cell death which is mediated by the exotoxin CpnT (Channel Protein with Necrosis-inducing Toxin). CpnT contains a C-terminal toxin domain named tuberculosis necrotizing toxin (TNT) that is secreted in macrophages and kills macrophages by depleting cellular NAD+. The discovery of CpnT challenged a long standing paradigm in the field that Mtb does not produce exotoxins and prompted many questions as to the biogenesis and export mechanism of such a toxin. Herein we have investigated the biogenesis, secretion, and enzymatic activity of CpnT in order to establish key steps in its trafficking pathway. We discovered that CpnT export from the Mtb cell depends on the ESX-4 system in coordination with the WXG100 proteins EsxE/EsxF. We found that EsxE/EsxF are pore-forming proteins which form an outer membrane channel for CpnT secretion. Our study is the first to directly observe pore-formation by a small Esx protein pair and we show that this is a general property of Esx proteins from M. tuberculosis. Following secretion, the translocation of TNT into the macrophage cytosol depends on the ESX-2 and ESX-4 secretion systems. We discovered that ESX-2 and ESX-4 are required for rupture of the mycobacterium-containing vacuole and trafficking of TNT to the cytosol in macrophages. Secreted TNT induces cytotoxicity via enzymatic hydrolysis of NAD(P)+ which activates the necroptosis cell death pathway. These studies establish a new paradigm for type VII secretion systems in the physiology and pathogenesis of tuberculosis.

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