The Proteasome of Mycobacterium tuberculosis Is Required for Resistance to Nitric Oxide

Abstract

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

“…The M. tuberculosis wild-type strain used was 1424, a derivative of M. tuberculosis H37Rv carrying a non-restrictive rpsL mutation conferring streptomycin resistance (23). The M. tuberculosis uvrB mutant strain MHD9 (11,12) was kindly provided by H. Darwin and C. Nathan. Cultures of M. tuberculosis were grown in OADC enriched Middlebrook 7H9 medium or modified Dubos (Difco) supplemented with albumin and 0.2% glycerol at 37°C in a rolling incubator at 2 rpm.…”

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

“…Mutants in the proteasome-associated genes mpa and pafA were more resistant to H 2 O 2 than wild type Mtb 19 and similarly, depletion of the proteasome resulted in increased resistance to oxidative stress (p=0.002 for PrcBA Tet-ON and p=0.007 for PrcBA Tet-OFF , Fig. 2c).…”

“…We next sought to determine the impact of prcBA silencing on in vitro growth and survival under nitrosative stress because previous studies predicted that the Mtb proteasome is essential for optimal growth on agar plates and required for defence against reactive nitrogen intermediates (RNI) 6,19 . Proteasome depletion impaired growth of Mtb on agar plates and, to a small degree, in liquid culture (Fig.…”

“…In contrast, prcA and prcB were predicted to be essential for optimal in vitro growth of Mtb 6 . Inactivation of two proteasome-associated genes, mpa and pafA, reduced Mtb virulence in mice, suggesting that the proteasome is also important for pathogenicity 19,20 . Mpa encodes a proteasomal ATPase, which is homologous to ATPases found in the regulatory cap of the eukaryotic 26S proteasome and likely involved in substrate recognition, unfolding and translocation into the proteasome core 15,21,22 .…”

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

“…Recently it has become clear that bacteria encoding a proteasome [9][10][11], like for example Mycobacterium tuberculosis (Mtb), use a similar tagging system termed pupylation [12][13][14]. In this process, the prokaryotic ubiquitin-like protein (Pup) is conjugated to substrate-lysines via its deamidated C-terminal GGQ-motif [12,14].…”