The goal of this research program is to characterize the biology of NadD (nicotinate adenylyltransferase) from Mycbobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). During infection, Mtb faces a diverse set of host microenvironments and host chemistries that can lead to proteotoxic stress, including hypoxia, mild acidity, metal starvation or intoxication, and reactive oxygen and nitrogen species. Antibiotic treatment of TB patients can damage Mtb proteins by stalling nascent proteins on the ribosome or via the formation of reactive oxygen or nitrogen intermediates. Many of these host stresses are associated with preventing or slowing of Mtb replication and the development of non-genetic, phenotypic drug resistance. Mtb maintains protein integrity with the proteostasis network ? a collection of over one hundred proteins, including chaperones and their cofactors (ClpB, DnaK, DnaJ1, DnaJ2, GrpE, GroEL/ES) and barrel-shaped proteases (ClpP1P2 and Mtb20S). The transcription of many members of the proteostasis network is induced by proteotoxic stresses such as heat. Genetic and chemical disruption of components of the proteostasis network is associated with decreased virulence in macrophage and murine models of TB. We sought to identify compounds targeting components of the proteostasis network by using a chemical screen for compounds whose activity was potentiated by a proteotoxic stress generated by heat. Our characterization of one heat-stress potentiated compound unexpectedly revealed NadD as the target. NadD is a high priority drug target that lies at a critical intersection of de novo and salvage pathways for NAD(H) and NADP(H) biosynthesis. Chemical inhibition of NadD in wild-type Mtb led to an increase in intrabacterial protein aggregation. We hypothesize that loss of NadD disrupts the function of NADPH-dependent antioxidant defense systems and/or NadD plays a role as an unconventional protein chaperone. This research takes two complementary directions:
in Aim 1, we will use genetics and chemical-biology to characterize NadD?s role in the proteostasis network, in particular by monitoring generation of intrabacterial reactive oxygen and nitrogen species, protein carbonylation, and protein aggregation; and in Aim 2, we will define the mechanism by which NadD contributes to Mtb proteostasis. These studies will lay the groundwork to understand how NadD contributes as a noncanonical member of the proteostasis network during infection and antibiotic treatment.
There is an urgent need to understand how Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), survives stresses of host immunity and antibiotic treatment. Mtb?s proteostasis network, composed of over a hundred proteins that maintain protein homeostasis via their refolding or degradation, plays a critical role both under nonstress conditions and during pathogenesis. In this proposal, we will characterize NadD, encoding a nicotinate adenylyltranferase (pyridine nucleotide biosynthesis), as a noncanonical member of Mtb?s proteostasis network.
