The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

Abstract

Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function.

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

“…The ppk-1 mutant strain was compromised in its ability to survive under nitrosative stress compared to the wild-type and complemented strains. Since polyP levels have been shown to regulate activity of Lon protease in E. coli, it is possible that polyP regulates the activity of the mycobacterial PrcBA proteasome system, thereby promoting degradation of misfolded proteins and providing amino acids for synthesis of new proteins critical for the tubercle bacilli to persist in the host (9,38,41,59). To evaluate the importance of polyP levels in survival of M. tuberculosis inside the macrophage, the growth kinetics of the ppk-1 mutant strain were compared to those of the wild-type and complemented strains.…”

“…The low efficiency of TB therapy has been attributed to multiple factors, such as noncompliance of patients, high bacterial loads, reduced metabolic rates of the organism, and the emergence of persisters or drug-tolerant bacteria due to their adaptability to the local microenvironment in the host lungs (3)(4)(5)(6). A number of pathways, including energy production, stringent response, trans-translation pathways, proteasomal protein degradation, toxin-antitoxin modules, efflux pumps, or switching to utilization of cholesterol and fatty acids as a carbon source have been implicated as contributing to M. tuberculosis persistence (7)(8)(9)(10)(11)(12)(13)(14).…”

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…These include dlaT (encoding dihydrolipoamide acyltransferase, a subunit of the pyruvate dehydrogenase complex) (209), menA, which is involved in menaquinone biosynthesis required for ATP (210), and cydC, which encodes a transporter for cytochrome bd assembly required for energy production during hypoxia and in vivo infection (211). Conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of M. tuberculosis in vitro, rendered bacilli more susceptible to nitric oxide, and impaired bacillary long-term survival during chronic infection in mice (212).…”

Mycobacterial Ubiquitin-like Protein Ligase PafA Follows a Two-step Reaction Pathway with a Phosphorylated Pup Intermediate

“…Conjugation of Pup to proteins can target them to the M. tuberculosis proteasome in vitro (13) and in vivo (14). Inhibition of the M. tuberculosis proteasomal system has gained attention due to its role in defending the bacteria against nitroxidative stress and its requirement for M. tuberculosis persistence in infected mice (15,16). Direct inhibition of the M. tuberculosis proteasome has been shown to be bactericidal in non-replicating M. tuberculosis (17).…”