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

Sabine Ehrt,Eric J Rubin,Sheetal Gandotra,Maria B Lebron

doi:10.1371/journal.ppat.1001040

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.

Highlights

Most cells continuously synthesize and degrade proteins in a regulated manner
The 20S proteasome belongs to the class of N-terminal nucleophile (Ntn) hydrolases, with a hydroxyl group of the amino(N) terminal threonine functioning as catalytic nucleophile that reacts with peptide bonds of substrates or the electrophilic functional groups of proteasome inhibitors [3]
The growth defects were restored in the complemented mutant. These data demonstrate that while the core proteasome is required for optimal growth of M. tuberculosis in vitro, it is not essential

Summary

Introduction

Protein degradation is highly selective and this is achieved in part by localization of protease active sites within a barrel-shaped complex. This self-compartmentalization was first discovered for the proteasome [1,2]. Bacterial proteasomes are only found in Actinomycetes [4], while other chambered proteases such as ClpAP, ClpXP, Lon, HslUV and FtsH are common in most bacteria [5,6]. Mycobacterium tuberculosis encodes a proteasome and two CLP proteases, but lacks homologs of Lon and HslUV [7]. The proteasome accessory factors, Mycobacterium proteasomal ATPase (Mpa) and proteasome accessory factor A (PafA), are important for defense against reactive nitrogen intermediates (RNI) and for virulence of M. tuberculosis in the mouse [8]. The M. tuberculosis 20S proteasome harbors electron dense plugs at the barrel ends created by the N-termini of its a subunits [11]

Methods

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Conclusion