Identification of a proteolysis regulator for an essential enzyme in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pup∼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup∼PanB in response to pantothenate levels. One Sentence Summary: A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .

Aldehyde accumulation in Mycobacterium tuberculosis with defective proteasomal degradation results in copper sensitivity.

Mycobacterium tuberculosis is a major human pathogen and the causative agent of tuberculosis disease. M. tuberculosis is able to persist in the face of host-derived antimicrobial molecules nitric oxide (NO) and copper (Cu). However, M. tuberculosis with defective proteasome activity is highly sensitive to NO and Cu, making the proteasome an attractive target for drug development. Previous work linked NO susceptibility with the accumulation of para-hydroxybenzaldehyde (pHBA) in M. tuberculosis mutants with defective proteasomal degradation. In this study, we found that pHBA accumulation was also responsible for Cu sensitivity in these strains. We showed that exogenous addition of pHBA to wild-type M. tuberculosis cultures sensitized bacteria to Cu to a degree similar to that of a proteasomal degradation mutant. We determined that pHBA reduced the production and function of critical Cu resistance proteins of the regulated in copper repressor (RicR) regulon. Furthermore, we extended these Cu-sensitizing effects to an aldehyde that M. tuberculosis may face within the macrophage. Collectively, this study is the first to mechanistically propose how aldehydes can render M. tuberculosis susceptible to an existing host defense and could support a broader role for aldehydes in controlling M. tuberculosis infections. IMPORTANCE M. tuberculosis is a leading cause of death by a single infectious agent, causing 1.5 million deaths annually. An effective vaccine for M. tuberculosis infections is currently lacking, and prior infection does not typically provide robust immunity to subsequent infections. Nonetheless, immunocompetent humans can control M. tuberculosis infections for decades. For these reasons, a clear understanding of how mammalian immunity inhibits mycobacterial growth is warranted. In this study, we show aldehydes can increase M. tuberculosis susceptibility to copper, an established antibacterial metal used by immune cells to control M. tuberculosis and other microbes. Given that activated macrophages produce increased amounts of aldehydes during infection, we propose host-derived aldehydes may help control bacterial infections, making aldehydes a previously unappreciated antimicrobial defense.